The tribulations of a stateless European child in his discovery of America and his thorny path to protein thermodynamics

G. Semenza and A.J. Turner (Eds.) Selected Topics in the History of Biochemistry: Personal Recollections VIH (Comprehensive Biochemistry Vol. 43) ~ 2004 Elsevier B.V.

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Chapter 7

The Tribulations of a Stateless European Child in His Discovery of America and His Thorny Path to Protein Thermodynamics SERGE N. TIMASHEFF

Professor Emeritus, Biochemistry Department, Brandeis University, 415 South St., Waltham, MA 02453, USA La Forza del Destino: Fate can make any man's life a succession of unexpected turns that must be taken and thorny hedges that must be crossed. Such is the lot of stateless people, even though their lives may never be threatened.

Origins I was born in Paris like were most Russian 6migr~s, or so it always seemed to me. My parents were refugees from the Bolshevik revolution of 1917 [1]. My father, Nikolai Sergueevich Timasheff, was a professor of jurisprudence at the University of St. Petersburg. When in 1921, it became evident that he would be a r r e s t e d and shot by the Lenin government, as was the fate of thousands of intellectuals, my mother and he set out on foot for Finland, with essentially just the clothes they had on. After two weeks of walking through forests and swimming across the Sestra river, they reached Helsinki. Meandering through G e r m a n y and Czechoslovakia, they finally settled in Paris where, on April 7, 1926, a major event occurred: I was

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born. As I recall, I was a delightful child and the joy of the whole family. The name Timasheff is of Tatar origin and I vaunt myself at being a descendant of Tamerlane, if not Genghis Khan. The clan comes from Astrakhan, close to the Caspian Sea, which was conquered rather late in history by Ivan the Terrible. I do c a r r y dominant Tatar genes: when at one point I tried to grow a beard, all that I could produce was a few tufts on my chin in true Fumanchu style. I am also lactose intolerant, which probably accounts for my total dislike of milk from early childhood, and my early propensity for red wine. A check of the genealogy three generations back from my paternal grandfather does not show any non-Russians, his wife, my grandmother, was 100% German, and Lutheran, and hardly spoke a word of Russian. On my mother's side, my grandfather's family were landed gentry from central Volga, the heartland of Russia. My grandmother, who hailed from Kiev, carried Polish and H u n g a r i a n blood, as well as that of some French Huguenots who escaped to Russia under the ruthless repression by Louis XIV. And so, my pool of genes is a little UN, which may account for my character traits of timidity and indecision. Going back in history, my grandfather, Sergei Ivanovich Timasheff, had been a cabinet member in the reform government of the great Stolypin. He held the portfolio of Commerce and Industry. His principal accomplishment was the institution in Russia of a social security system. Sometime after Stolypin's assassination, he was called in by the Emperor. W h e n he entered the study, Nicholas II embraced him and, with tears in his eyes, told him that he was being dismissed from his post, as he bestowed on him one of the greatest honors of the Empire, the title of "State Secretary of his Majesty:.' To his question: "Your Majesty, why?" The answer was: "It must be so" My grandfather systematically had disdainfully rebuked all requests from Rasputin to appoint his friends and cohorts to posts in his Ministry. Later, in 1915, when the Russian army was experiencing a drastic shortage of ammunition, he was named to the

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newly formed committee on military procurement, as president of the Artillery Commission, working in close collaboration with the newly appointed, very talented War Minister, General Polivanov. By 1917, they had accumulated a stock of ammunition that greatly exceeded that of the Germans [2-4]. In the spring of 1917, a major offensive commanded by Generals Alexeeff, Head of the General Staff, and Ruzsky, Front Commander, was to be launched on the Northwestern Front. (These generals belonged to the young generation that had been formed at the General Staff Academy. Admission was by competition (exams) and family origins did not matter [4]. Alexeeff was of modest background, as was Brusilow, who inflicted a major defeat on the Germans in 1916. Denikin, Front Commander, was the grandson of serfs.) The offensive was slated to carry all the way to Berlin and, later, Lfidendorff said that the Germans could not have stopped it. Fate decreed otherwise. Total mismanagement by Nicholas II, under pressure from the Empress, whose advisor was Rasputin, led Russia into revolution in February 1917 and finally to Lenin's coup d~tat. My grandfather died in 1920 in a Bolshevik prison, as did one of my uncles. On my mother's side, my grandfather, Nikolai Pavlovich Ruzsky was an industrialist. He was also a virtuoso cellist and, in the privacy of his St. Petersburg home, played trios and quartets with renowned musicians of the time, a number of whom were his personal friends. His first cousin, to whom he was very close, was General Nikolai Vladimirovich Ruzsky, who very early in the war defeated the Austrians and took Lvov. He was to command a major thrust of the final offensive. Instead, he got the agonizing experience of Nicholas II coming to this headquarters, where after a few days of hesitation over what action to take to crush the revolution, the Emperor was forced by delegates of the Duma to abdicate the throne [2,3,5]. Later, when the army's comm a n d fell to soldiers' revolutionary committees, General Ruzsky retired to the Caucasus. There he was approached to join the newly formed Red Army. When, pointing to his epaulettes, he refused, he was jumped upon by the mob and trampled to

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death. (General Polivanov did accept the call from Trotsky, a n d it is he who built the Red A r m y [3-5], even t h o u g h legend credits Trotsky with t h a t feat. Polivanov h a d betrayed his oath a n d dishonored his epaulettes.) My m a t e r n a l grandfather died in Paris. My father lived a long life p u r s u i n g scholarly studies until close to the end. W h e n he died, he left as heritage 18 scholarly books. My m o t h e r died of cancer six m o n t h s before I received my PhD.

"Little Russia" in P a r i s We lived in Chaville, a m o d e s t suburb of Paris. Fitting the name, it h a d an innumerable population of cats. O u r life was divided between being members of the Russian c o m m u n i t y a n d p a r t of the host country. France was not a melting pot a n d most Russians expected to r e t u r n home after the wished for downfall of the c o m m u n i s t regime. All h a d the status of "Stateless" a n d lived with N a n s e n passports, which the League of Nations h a d created for Russian a n d A r m e n i a n refugees. French citizenship was not easy to obtain a n d not m a n y were seeking it. Nevertheless, w h e n war came, the young m e n were i n d u c t e d into the army. Years later, while w a n d e r i n g in the Russian cemetery outside Paris, I r a n into the grave of a m a n w h o m I r e m e m b e r as a star Sokol. He was killed in Algeria. At the time, there was a large Russian colony in Paris a n d suburbs, some estimate it at more t h a n 100,000. There was a very active Russian cultural life focused locally a r o u n d the Orthodox parishes t h a t h a d s p r u n g up a r o u n d Paris, a n d centrally on Paris, a n d a variety of youth organizations, from Russian boy scouts to Russian Sokol, a pan-Slavic organization t h a t concentrated on gymnastics a n d which h a d seen birth in Czechoslovakia prior to World War I. Emigr~ artists periodically p r o d u c e d Russian classical plays a n d r e n o w n e d musicians gave major concerts, which we attended. A n d so, I, along with thousands of others, led a double life: we went to French school, a n d

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played w i t h our very disciplined F r e n c h schoolmates on one hand, and, on the other hand, we gathered w i t h our more e x u b e r a n t Russian playmates u n d e r the close supervision of p a r e n t s b o t h for play and for instruction. For all the children, b o t h Russian and French, World War I was a vivid reality. We h a d all "lived t h r o u g h it," even t h o u g h we were b o r n almost a decade after its end. The Russian children h a d "also lived through" the Russian Revolution, Civil War, a n d the famine of 1920 created by Lenin's violent war against the peasants. So horrible were all these events t h a t they h a d indelibly m a r k e d society and left their s t a m p on our p a r e n t s for their lives. In France, Thursday was a school-free day. It became logically the day for the "Russian school:' We were t a u g h t Russian history a n d literature. O u r teachers were some of the mothers, m a n y of w h o m were highly educated. Invariably, one of our preceptors was a Russian army colonel who led us in calisthenics a n d outdoor games, a n d lectured us on Honor, Duty, a n d Patriotism. Each year, every p a r i s h a n d organization h a d its feast at which the Russian young performed staged scenes from the Russian literature, e.g., Pushkin's Boris Godunov, recited poetry, d a n c e d characteristic Russian village dances, a n d sang in chorus Russian folk songs, as well as P u s h k i n a n d L e r m o n t o v historic poems set to music by great Russian composers. The principal event of the year was the Day of Russian Culture, which brought together u n d e r one roof anti-Soviet Russians of the most diverse political inclinations. It was presided by the G r a n d Duke a n d Metropolitan Evlogii, who h a d been a p p o i n t e d by Patriarch T i k h o n ( m a r t y r e d by the Bolsheviks in 1924) as head of the Russian Orthodox Churches i n W e s t e r n Europe. He was a m a n of great moderation respected by everyone. In Russia, he h a d been an elected m e m b e r of the Duma. He was very attentive to children. I r e m e m b e r one day w h e n he was sitting on the sofa of our Chaville apartment, as usual, he took me on his knees. He t h e n gave me a sip of vodka from the tumbler t h a t my m o t h e r had just served him. My m o t h e r obviously was not pleased with this, b u t could say

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nothing. I was delighted. He t h e n shook his finger at me a n d said: "Now, remember, never d r i n k m u c h of this" I was seven or eight years old, and a rule h a d been established. His greatest achievement was the creation of the St. Sergei Theological I n s t i t u t e which assembled Russian theologians, philosophers, a n d historians of diverse views a n d quickly became world renowned. R e t u r n i n g to the great gathering, it took place in an indoor stadium. It o p e n e d w i t h a parade of the youths' organizations, led by the military cadets in u n i f o r m who carried the colors of one of the famous Imperial G u a r d regiments. At the tail end came the girls from the various ballet schools. In between were scouts, Sokol, a n d others. We stood at attention as the F r e n c h a n d Russian flags were raised to the playing of the Marseillaise a n d the Russian national anthem. We h e a r d speeches about love of one's country, loyalty a n d honor, a n d t h e n watched the selected best perform. There were choirs a n d balalaika a n d military orchestras. The best girls from the ballet schools d a n c e d numbers from the classical ballets. A n u m b e r of t h e m later became p r i m a ballerinas in various major ballet companies. The Sokol p u t on spectacular numbers on single a n d double bars, etc., which finished with a six story high h u m a n p y r a m i d at the top of which were b r a n d i s h e d Russian a n d F r e n c h flags: still the great alliance! S u m m e r s t a r t e d on July 14, the great national holiday which c o m m e m o r a t e s the mob's t a k i n g of the Bastille, an old fortressprison slated for demolition by the Ancien Regime, a n d the liberation of the Marquis de Sade and a few other "victims of persecution" School h a d just ended. After watching the fireworks, this m e a n t d e p a r t u r e to S u m m e r camp. I was with the Scouts, I was a cub. One evening h u n d r e d s of us assembled at Gare de Lyon, b o a r d e d trains, a n d off we were for w h a t was to become later t h e F r e n c h Riviera. There were six to eight of us per c o m p a r t m e n t , plus one adult. All our baggage (mainly backpacks) were p u t on the floor between the two b a n k s a n d we were "put to bed"." two in the baggage nets overhead, the rest on the b a n k s a n d on the baggage. The locomotive gave its whistle

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and with heavy puffing s t a r t e d off. Between Paris and Marseille, locomotives were changed five or six times. The noise, smoke, and banging were most exciting for the kids. We were ordered to sleep, so we kept quiet. On arrival in the morning (Marseille, Toulon, or Cannes, depending on where the camp was located), we first boarded buses and finally m a r c h e d to the camp. Camps were established on plots of land rented from some farmer. This land was covered with the magnificent M e d i t e r r a n e a n pines. Each troop was t a k e n to its designated location which had been cleared of brush. Next, the tents were set up and ditches were dug around them, as thunderstorms can be violent on the Mediterranean. We eight to ten year olds performed such tasks as assigned. We filled our bags and pillowcases with straw, lined them up inside the tents and covered t h e m with the blankets t h a t each one had brought from home. We were now settled for between four and eight weeks. I was in total amazement when a few years later I arrived at a boy-scout camp in the USA. The campsites had tents set on raised wooden platforms. There were bunks, mattresses, pillows, and blankets. Like in boy-scout camps anywhere else, life was by the bugle. We did calisthenics, bathed on the beach, went on hikes, cleared u n d e r b r u s h as a fire-preventive measure, sat by campfires, sang, played games, etc. We also had sessions on Russian history and culture. The soil was dry and rocky; there were snakes and scorpions. Nevertheless we always walked barefoot. Scorpions were easy to find: one just had to lift a few stones. I do not recall any victims. At the time, the "Riviera" had not been discovered by tourism. It was a series of craggy capes coming down abruptly into the aquamarine water with sandy coves in between, a sight of incomparable beauty: St. Tropez was a fishing village, Cannes was a small town with a beautiful golden beach. It was a little paradise, now lost, never to be regained as it stands desecrated by the continuous line of hotels and other tourist accommodations, a never-ending traffic jam, and the loud noise of people "enjoying a vacation" Gone are the vineyards, melon plantations, and pine trees. The last mostly b u r n e d

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down with the a p p e a r a n c e of tourists; there were no fires in my childhood.

Insertion into French Society A major effort was made to preserve the Russian culture in the children, while i n t e g r a t i n g t h e m into everyday F r e n c h life. There was no contradiction in this, as nationality was highly r e s p e c t e d in France a n d the French, in general, liked their Russian guests, whose great grandchildren, now fully French, are keeping to this day m a n y Russian traditions. In this society, preschool life was almost exclusively Russian, insertion into the host c o u n t r y c o m i n g with school. A n d so, the dramatic day came when, at the ripe age of eight, I h a d to confront F r e n c h school. I resisted physically all the way as my m o t h e r dragged me along the street. I obviously cherished freedom. At that point, I could read and write Russian, I could add, subtract, a n d multiply. I k n e w a fair a m o u n t of geography for which I h a d a fascination. My knowledge of F r e n c h was most rudimentary. I have a vivid m e m o r y of sitting with my back to the class and sulking. At the end of the day, the elderly teacher offered to play a game w i t h me; I graciously accepted her offer a n d one h o u r later I h a d been t a u g h t how to divide. F r o m t h a t m o m e n t on, school was my love a n d each day opened new horizons to my life. It was not long before my personal historical origins expanded to Clovis, Charlemagne, a n d J e a n n e d~rc, who easily merged w i t h Alexander Nevski, Ivan the Terrible, a n d Peter the Great, as later would George Washington a n d A b r a h a m Lincoln. On only one personage was there no compromise: Napoleon. To the F r e n c h he was a great hero, to this Russian infant he was a great villain. I could tell step by step his p l u n d e r i n g invasion of Russia in 1812, the b u r n i n g of Moscow, a n d his defeat by the Russian people, as in the mild late fall of 1812 he a b a n d o n e d the r e m n a n t s of his army a n d fled across the Berezina to be

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followed by Cossacks who in 1814 c a m p e d on the C h a m p s Elys6es. This is the only time w h e n I challenged a teacher in class, which caused my m o t h e r to have a talk with the Headmistress. In school my great loves were mathematics, geography, a n d history, particularly military. After five years of small s u b u r b a n Catholic schools, I was accepted (by entrance exam) at the Lyc6e Hoche in Versailles. It was 1938 and I was 12 years old. There were 35 of us in class, r a n g i n g from 12 to 18 year olds who were waiting to enter military service. The discipline was strict. We rose w h e n t h e teacher entered t h e room a n d stayed silent a n d attentive to the lesson. We did not r u n in the corridors a n d t a l k e d in low voices. B r e a k i n g of these rules brought on the p u n i s h m e n t of having to report on Sunday for a prescribed n u m b e r of hours, d u r i n g which one copied an assigned n u m b e r of pages from a text book. One day I was caught r u n n i n g a n d spent the Sunday in this pastime. The c u r r i c u l u m t h a t year consisted of F r e n c h literature, French composition, history, geography, geology, English, German, m a t h e m a t i c s (algebra a n d w h a t I later identified as t h e o r y of numbers), a n d drawing. Classes s t a r t e d at 8 a.m. (I had to take the 7 o'clock t r a i n every morning) a n d e n d e d at 4 p.m. There was always 2-3 h w o r t h of homework. As there was no school on Thursday a n d Saturday afternoon, on those days the lyc6e offered supervised recreation which could consist of a teacher giving a 2-h lecture, illustrated w i t h slides, on his s u m m e r travels in the S a h a r a or in Indochina, or of a n accompanied visit to a museum, a castle, or a cathedral, all with detailed explanations. It could also be a t t e n d a n c e at a Moliere or Racine play at the classical t h e a t e r in Paris especially organized for school children. Those who went absorbed avidly w h a t was offered a n d t h e n frequently a r g u e d among themselves about details. Study was t h o r o u g h a n d the examinations serious. After an exam, the grades were read out aloud in descending order a n d it was a n n o u n c e d where the cutoff was set. The grading basis was 20. A grade of 13 was excellent a n d a 14 called for felicitations in front of the class.

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(Later this p r e s e n t e d a problem to the High School Principal in Cambridge, MA, who on seeing my average, 13+/20, n e e d e d to be p e r s u a d e d by my father to accept me even on probation.) I still recall two of the final exams which I took at the age of 13. In history, a single question 2 h exam: "Compare a n d contrast the Byzantine a n d Arab cultures"; its c o u n t e r p a r t in geography was: "Indochina: draw its map, w i t h rivers, mountains, towns, etc. a n d discuss thoroughly its physical a n d economic (natural resources, etc.) aspects" In all subjects any spelling error or crossed-out word on the exam paper lowered the grade by as m u c h as one point. All w r i t i n g h a d to be in ink, using a p e n a n d inkwell. We read F r e n c h classical literature as assignment, a n d for e n t e r t a i n m e n t we read Fenimore Cooper, Jack London, M a r k Twain, a n d Edgar Allan Poe, along w i t h Jules Verne a n d Robert Louis Stevenson. This schooling has d i s a p p e a r e d long since, as it has been replaced by "relevant" subjects.

War: "Drole de Guerre" In Paris, my father worked as one of the editors of a middle of the road Russian daily newspaper. He followed diligently the development of the Soviet Union a n d later of Nazi Germany. He despised a n d loathed b o t h regimes a n d m a n y of his articles on the editorial page bore on these systems. He is one of a few who had read all of Marx, all of Lenin, a n d Mein Kampf. The last, he qualified as the most boring book t h a t he h a d ever encountered. Years later, w h e n he was at Fordham, he was occasionally invited to participate in Catholic-Communist debates. He was highly successful at destroying the Communists' arguments by direct quotations from M a r x a n d L e n i n which their faithful New York followers h a d obviously never read. M a r x h a d made a n u m b e r of anti-Semitic declarations. As usual, everything has its anecdotal side: the best way to follow Soviet developments, from the building of new factories to new persecutions of the Church, was to read their newspapers, if one

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knew how. Therefore, for some time, my father subscribed to Izvestie and Pravda. This brought frowns of suspicion, fortunately not from people of authority. One day, when he and Professor Baerwald, a G e r m a n anti-Hitler Catholic, gave talks at a symposium on questions of democracy, one of the written comments was: "One speaker was a Communist, the other a Nazi, so what are we to believe?" In 1936, my father received a totally unexpected offer for a one-year visiting professorship from H a r v a r d University. He went. Every summer he came back to his family in Chaville. As this was renewed annually, he decided that my mother with the two children had to move across the Atlantic. In April 1939, c o n t r a r y to the general consensus, he predicted that World War II would start on September 1 and bought us tickets to sail on the "Normandie" in late August. His reasoning was as follows: Hitler had made territorial demands on Poland from which he would not back down, Great Britain had g u a r a n t e e d the frontiers of Poland: there could be no compromise. Historically, G e r m a n y had s t a r t e d its wars before the bringing in of the harvest; September 1 was a Sunday, a day on which the British government would be sound asleep. A n d so at the hour of five o'clock tea, Hitler marched into Poland. The French, for security, kept the "Normandie" in New York, where eventually it b u r n e d down and we were stranded. France entered the war the next day after Britain. I was 13 years old and found this very exciting. This was not the view of my mother and my aunt, both of whom had been frontline nurses in the Russian army during the First World War. Their field hospital was closest to the front and it is there t h a t the wounded were delivered first. Their hospital was constantly bombed, and the entire staff received the Order of St. George, the military medal of imperial Russia. They had been close observers of the horrors of war and were appalled by the enthusiasm of some French women or men, too old to serve in a second war, for this new chance to beat "les boches" This enthusiasm was not shared by m a n y of the young men who were being

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mobilized. I recall, in our little suburb, a young m a n being dragged by some women to the reporting center. To his loud protests, came the reply: "You will be shot if you don't report" Mobilized soldiers were requested if possible to bring their own boots, blankets, etc. Such was the preparedness of France after being governed by the Front Populaire. France, which in early 1939 had started a r e a r m a m e n t program, had better t a n k s t h a n Germany. A French colonel, by the name of Charles De Gaulle, had w r i t t e n a book on how to use t a n k s in mobile warfare. Like any theory, it was verified in the spring of 1940 by General Guderian. The one t h i n g t h a t was lacking was the will to fight. I recall when one day I was bringing the newspaper home, I was stopped by two soldiers who asked to look at it. When they saw how far and fast the G e r m a n army was advancing in Poland, they looked up and, with smiles, said: "Good, soon this will be over and we will all go home" Of course, the friendship treaty between the Soviet Union and Hitler did not induce the communists on the front to put up excessive resistance. A n d so, it should have been no surprise when, in J u n e 1940, France was overrun by G e r m a n t a n k s in a matter of six weeks. The a p a r t m e n t house in which we lived overlooked the electric rail line from Paris Invalides to Versailles. A sport which kids practiced, including this one, was to jump on the g u a r d of the third rail with both feet, r u n along it, and jump off. This did not delight parents, but the sternest measures did not act as deterrents. I am not aware of any casualties. Under our windows there was a small siding. This is where freight cars would be brought by a locomotive (and I was a great fan of locomotives) with supplies for the Villacoublay air base located 5 k m away. We often took walks there through the woods. In those days, the planes landed on a flat grass-covered field. Also, in the 1930s people looked up every time they h e a r d a plane and identified it as a monoplane or a biplane. Once every year there was the great show in which planes performed all sorts of acrobatics and gyrations in the air. This was followed by fireworks at night, as was normal for holidays in France.

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As war was declared, an anti-aircraft g u n was brought to this siding a n d from our windows we could see h a l f a dozen soldiers sitting bored on the doorstep of the barrack t h a t h a d been p u t up. Frequently, some would just walk away from their post a n d w a n d e r a r o u n d Chaville. The two t h a t h a d expressed their delight at the collapse of Poland belonged to this unit. By government order, all windows got t a p e d a n d people were encouraged to concoct their o w n gas masks, which consisted of gauze stuffed w i t h some absorbing minerals. A blackout was imposed. A few days after the start of w h a t the F r e n c h referred to as "drole de guerre:' since not one artillery shell was exchanged b e t w e e n the M a g i n o t and Siegfried lines, the sirens s o u n d e d off: air raid alarm! It was already dark. People r a n out of the houses, carrying their m a k e s h i f t gas masks. Each block h a d air-raid wardens whose duty was to see to it t h a t the blackout was not violated. As the crowd gathered outside our apartm e n t building, the w a r d e n saw two brightly lit windows. There followed blasts from his whistle, t h e n swearing. Finally, he s t o r m e d into the house a n d 2 m i n later the windows became dark. He t h e n reappeared, with a s h r u g of the shoulders: "m..., it was my own apartment:' The French "je m e n fous" once again h a d prevailed. This is, in fact, the attitude t h a t has p e r m i t t e d France to muddle t h r o u g h decades of its history. Half an h o u r later there was a beep from a siren somewhere a n d people went back to bed, only to be awakened by the "all clear" siren 3 h later. The next day r u m o r s circulated that the G e r m a n s h a d destroyed t h r e e cities in the N o r t h of France. In fact, no G e r m a n plane h a d crossed the frontier. The whole t h i n g h a d been simply a greater Paris practice run.

Sailing away to the U S A Now, we were in Paris a n d h a d to travel to America. With the start of the war, the F r e n c h Line stopped functioning a n d the only one t h a t c o n t i n u e d was the US Line. However, now n o t h i n g

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sailed from Le Havre, the p o r t of d e p a r t u r e was Bordeaux. The US Line agreed to accept our N o r m a n d i e tickets as p a y m e n t a n d told my m o t h e r t h a t we would sail w i t h i n t e n days or so. This was w a r t i m e a n d all travel h a d to get authorized. A few visits by my m o t h e r to the Prefecture p r o d u c e d a d o c u m e n t which p e r m i t t e d our travel to Bordeaux, gave residence there for 15 days a n d prohibited a r e t u r n to Paris. We h a d to go to Bordeaux with a m o u n t a i n of t r u n k s , this being a definitive move from one c o u n t r y to another. All of this h a d to be transferred from Chaville to Gare d:Austerlitz in Paris. There was one complication. In their wisdom, the F r e n c h government, recalling how in 1914 General Gallieni h a d t r a n s p o r t e d by Paris taxis an army from Paris to the flank of the G e r m a n army positioned on the Marne, requisitioned the Paris taxis. In 1914 this led to victory in the first battle of the Marne. Now they were p r e p a r e d to win the t h i r d battle of the Marne. I r e m e m b e r seeing taxis stored in a n open field where, eventually, they r u s t e d away. My uncle h a d a small car, but this was far from enough. Somehow he found someone with a t r u c k a n d the whole load was t a k e n to the railway station. My uncle, being a m a n of the world, u n d e r s t o o d well how to handle certain situations. He properly rewarded t h r e e porters, the ticket controller, a n d the head c o n d u c t o r on the train. I recall how, w h e n we went t h r o u g h t h e control, the lead p o r t e r called out: "Three sleeping compartments:' The baggage was loaded into an e m p t y c o m p a r t m e n t a n d the next morning, w h e n we arrived in Bordeaux, the head conductor, being a m a n of his word, lived up to the bargain: he p r o d u c e d t h r e e porters who took us to a large hotel close to the railway station. Bordeaux, the refuge of fugitive F r e n c h governments whenever G e r m a n s advanced, was t e a m i n g with F r e n c h who h a d left Paris (although m a n y h a d gone to their villages), people like us w i t h i m m i g r a t i o n visas to the U n i t e d States or some other places a n d "rich Americans" who h a d become s t r a n d e d as the war broke out. A view of the recent F r e n c h second-rate film "Bon Voyage" brought back m a n y memories of the chaos,

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although the film referred to J u n e 1940 w h e n the G e r m a n s were advancing. A visit to the US Lines office confirmed t h a t a ship was, indeed, arriving. It came a n d left h a l f empty. The US Congress had passed the Neutrality Act, which, a m o n g other things, forbade non-American citizens to be t r a n s p o r t e d on A m e r i c a n ships. The s t r a n d e d A m e r i c a n tourists did not sail, because, as they were telling, they k n e w that the US governm e n t would send a ship to rescue them. Then, why pay for the passage? Eventually the US government did comply a n d they all left at taxpayers' expense. Life was guided by rumors: is a ship coming or not? Someone h a d discovered a n inexpensive Greek restaurant, "Satiros:' A n d a whole crowd, immigrants, as well as "rich Americans" found itself fluctuating between the "Satiros" on days w h e n the news (rumor) were bad (there was no ship), a n d the "Grand Hotel" d i n i n g room w h e n d e p a r t u r e seemed imminent. For us an insoluble d i l e m m a set in. We h a d been in Bordeaux for 15 days. We were allowed neither to stay there any longer nor to r e t u r n to Paris. The situation was resolved in good French style. My m o t h e r went to the Prefecture to ask for an extension of our stay in Bordeaux. The official refused a n d told her to go to Paris to extend a p e r m i t t h a t she h a d obtained there. W h e n it was p o i n t e d out to h i m t h a t a r e t u r n to Paris was forbidden by the Paris Prefecture, his answer was: "Madame, t h a t was Paris; here we are in Bordeaux." As she walked out of his office in total frustration, a g e n d a r m e caught up w i t h her a n d whispered "Madame, p u t a 20 franc note into your passport:' Back she went, following this advice, a n d faced the same official. He took the properly stuffed passport, examined it a n d said: "Madame, here in Bordeaux, o n se l o u t of w h a t they do in Paris:' He s t a m p e d the paper a n d extended our p e r m i t to stay. After a few more days, our d e p a r t u r e was settled. A liner, t h e USS Washington was c o m i n g a n d the US Congress h a d modified the law, p e r m i t t i n g foreigners with i m m i g r a t i o n visas a n d a relative resident of the USA to sail on A m e r i c a n ships. We fitted this criterion. My m o t h e r went to the US Line office.

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The a t t e n d a n t took the "Normandie" tickets a n d said: "There is a 50 (1939) dollar supplement in cash per person" My m o t h e r h a d the funds, as my father h a d sent over to her a good p a r t of his savings. She p u t $150 on the table, he took t h e m a n d issued the tickets. No receipt was issued for the cash. We h a d a cabin for t h r e e persons: it t u r n e d out to be a two-berth cabin to which a folding cot h a d been added. A n d so we could sail. My m o t h e r went to the post office to telegraph my father about our departure so t h a t he could meet us in New York. But, this being wartime, there was strict censorship a n d secrecy. Finally, they accepted the message: "Leaving Tuesday:.' My father on receiving this in Cambridge, MA, picked up the NY Times a n d looked in the section on sailings. He found the listing of the USS Washington leaving Bordeaux on the proper Tuesday at a specifled hour a n d its exact arrival time in New York. So m u c h for w a r t i m e secrecy! Now, things were easy. This was a r e g u l a r trans-Atlantic liner. The m o u n t a i n of baggage was picked up at the hotel a n d registered for t r a n s p o r t a t i o n in the hold of the ship. We saw it next in New York, where the customs official proceeded to searching everything in great detail. We b o a r d e d the s t a n d a r d boat t r a i n a n d proceeded to Le Verdon, which is Bordeaux's deep-sea port. This being war, France h a d i n s t i t u t e d tight control on t a k i n g money out of the country. My mother, however, was in possession of my father's savings. To avoid confiscation, she sent it to my g r a n d m o t h e r in Paris who r e m a i n e d there t h r o u g h o u t the war with my a u n t and other relatives. The t r a i n got to Le Verdon a n d we got off. There were several barriers to pass: passport control, customs control, a n d money control. At each point there were m e n in uniform. In t r u e F r e n c h fashion, war or no war, nobody checked anything: we could have been without passports and we could have t a k e n millions in cash. W h e n we b o a r d e d the USS Washington, US officials did properly check passports a n d i m m i g r a t i o n visas. The USS Washington stayed m o o r e d for two days. T h e n anchor was lifted and, without any whistle or horn, we sailed. The first day I could see a large line of ships on the horizon: we

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were following a convoy. Then, as we got away from the French shore a n d French waters, off we went on o u r own. The ship h a d huge A m e r i c a n flags p a i n t e d on its sides a n d a huge flag was flying from the mast. At night all this was illuminated by floodlights. One night we woke up feeling t h a t the ship had stopped. Next day, some passengers pried out from crew members the information t h a t we had been stopped by a G e r m a n s u b m a r i n e which was looking for a possible evacuation of the French gold reserve. W h e n they assured themselves t h a t it was not aboard, they left politely. We sailed on a n d I became acquainted w i t h life on a trans-Atlantic liner which, m u c h later, I enjoyed on several crossings on the occasion of sabbaticals. Finally, the famous silhouette of the New York sky scrapers appeared. We sailed past the Statue of Liberty and docked at a pier on the H u d s o n River. It was October 12, 1939 and, as I got off the ship, I discovered the New World together with Christopher Columbus.

"Clash of Civilizations" After one day in New York gazing at sky scrapers, I arrived in Cambridge, MA, where I was to live the next year. I was enrolled into the High a n d Latin school as a f r e s h m a n a n d came to face a "clash of civilizations:' I did not speak a word of English, b u t I k n e w English g r a m m a r by h e a r t a n d I could not u n d e r s t a n d the exercises t h a t were h a n d e d out in which incorrect expressions h a d to be corrected (e.g., t h e m for those, ain't, etc.). I k n e w all the rules about shall and will, who a n d whom, etc. (rules which, as a m a t t e r of fact, are totally broken these days even in serious writing). I could do simple translations (I had four years of English in France). The teachers were very k i n d to me: they used the few words of F r e n c h they could muster. At first, I was not required to do any class work; I was just to listen. To my classmates I was a curiosity; who was this zoo specimen t h a t h a d landed a m o n g them? I became k n o w n as "Frenchie:' a n d whenever I insisted t h a t I was Russian and not French I was

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told a d a m a n t l y that one's nationality is d e t e r m i n e d by the country where one was born. The contrast was overwhelming. I was lost in a school t h a t lacked the strict discipline to which I h a d been accustomed. The students, although quiet in class, were, by my criteria, a totally u n r u l y lot. I was flabbergasted by students d a r i n g to i n t e r r u p t a teacher with questions d u r i n g class. The sight of my classmates working w i t h pencil a n d eraser d u r i n g tests was beyond belief. I c o n t i n u e d t u r n i n g in beautifully inkw r i t t e n test papers without ever a correction. Little by little the English came. In this, Latin was of great help because I understood it well. I was so advanced in algebra, t h a t in exams with choice, I did all the problems to the dismay of the teacher (actually I had never u n d e r s t o o d that there was a choice). The most difficult subject to me was the history of Cambridge, MA, which did not fit into my scheme of world history. In fact, A m e r i c a n history was to me a difficult subject. I could not u n d e r s t a n d the i m p o r t a n c e of local wars, such as the French a n d I n d i a n War. To me, they were just colonial skirmishes as parts of major wars between E n g l a n d a n d France in the 18th century. Finally, I u n d e r s t o o d t h a t this was a different history. It was the history of a newly developing nation, the settlement of open territory, the g r o w t h of institutions, a n d not t h a t of the rivalries between long-established dynasties in lands settled for m a n y centuries by people of a generally uniform, Christian, culture. My new playmates m u s t have been also distraught by this little "Lord Fauntleroy" t h a t h a d l a n d e d a m o n g them. My t a l k about Hitler a n d Mussolini h a d t h e m amused: they did not quite know what it was all about, a n d most of t h e m could not find Poland on a map. By Christmas time I was b e g i n n i n g to get along in English, a n d by the end of the school year I could do all the work along with others. This, I m u s t stress, h a d not required the nowadays so p o p u l a r bilingual school; i m m e r s i o n did it. As s u m m e r came, my parents sent me off to a s u m m e r boy-scout camp to help my integration. W h e n I came back, my education h a d been complete a n d my vocabulary was greatly enriched, including m u c h that my parents wished I h a d not learned.

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In less t h a n a year I h a d become trilingual, without any effort on my part. I could have spoken four languages, were it not for my lack of self-discipline. Later, w h e n I was at Fordham Prep., my father, who h a d lectured in five languages, offered to teach me German. I was t a k i n g G e r m a n as the foreign language. On Sunday afternoons, we would go for a 1-h walk along the Bronx River. The conversation was in German. My father painstakingly corrected me. I was m a k i n g good progress. But then, I s t a r t e d finding all sorts of other t h i n g s to do. The walks became rarer a n d finally stopped. As a consequence, I c a n n o t read "Max u n d Moritz" in the original. In America there was no "Little Russia:' In the Boston area, the n u m b e r of ~migr~s was small. I acquired as playmates children of Russian professors at the various universities. They were being brought up as solely Americans, to the extent t h a t some did not know any Russian. Later they reproached their p a r e n t of having deprived t h e m of the possibility to read "War a n d Peace" in the original. In NewYork, there was some cultural activity, but not for the young. This c h a n g e d later with the arrival in the early 1950s of the large Second Emigration which settled principally a r o u n d New York a n d San Francisco a n d rapidly blended with the First Emigration b o t h in America a n d in Europe. A small window did get opened in 1941-42, w h e n Alexandra Tolstoy, the daughter of the great writer, founded the Tolstoy Center on Reid Farm, an estate 30 miles from New York t h a t was donated to the Tolstoy Foundation that she h a d just established together with Tatiana Schaufuss. They became friends w h e n the two of t h e m met in the L o u b i a n k a prison. Mrs. Schaufuss was a w o m a n of infinite energy and an iron will. The idea of Alexandra Tolstoy was to establish a center where Russian intellectuals would come in peace for weeks at a time to do scholarly studies on Russian culture a n d promote it in the USA. Her d r e a m was never realized, although large archives h a d been gathered, mostly by bequests. It t u r n e d out t h a t Mrs. Schaufuss was a Kiev childhood friend of my mother, who set out to help organize the children's camp in this new

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Russian beginning. T h a t first summer, the camp, where I spent two months, h a d as preceptors one of the ladies from Chaville a n d one of the Paris colonels who h a d e m i g r a t e d to America. In calisthenics, the old colonel could outdo any of the young kids. I vaguely r e m e m b e r t h a t s u m m e r a little girl who every day practiced ballet u n d e r her mother's supervision. She was Svetlana Beriozova; little did I k n o w that I was seeing a f u t u r e p r i m a ballerina w i t h the Royal Ballet. My father, in addition to giving fund-raising lectures, spent t h a t s u m m e r developing p a r t s of the vegetable garden, a n d the plot of strawberries, which w i t h h i m was a great passion. Later, the Tolstoy Foundation became one of the m a i n organizations for b r i n g i n g refugees from c o m m u n i s t countries into the USA, Australia, a n d other places: these included Tibetans, Vietnamese, as well as various East Europeans. Today the Center exists: it concentrates on giving poor children in Russia the footing necessary for a start in life.

The Jesuits

In the fall of 1940, my father obtained a p e r m a n e n t faculty position at Fordham, a n d so I was enrolled at F o r d h a m Prep., leaving the campus 11 years later w i t h a PhD. This school was more to my liking. There were 30 of us in the class. The school building was 100 years old. It h a d creaky wooden floors. But this did not prevent us from getting a n excellent education. The Jesuits k n e w how to impose discipline a n d their pedagogical skills were unsurpassed. A m o n g other things, they t a u g h t us to write clearly, concisely, a n d in an attractive style by i m i t a t i n g various classical authors. They t a u g h t us to speak clearly a n d logically in front of others. Diction a n d enunciation were emphasized. Elocution was p a r t of the curriculum. The F o r d h a m motto is Sapientia et Doctrina (wisdom a n d knowledge). This p e r m e a t e d down to the Prep. The school was m a r k e d by highly devoted teachers, b o t h Jesuits a n d laymen. We were

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given knowledge in a clear, systematic way, as the teachers endeavored to keep us interested a n d attentive. We were offered a well-rounded education, based on more t h a n two millennia of continually developing culture, Greco-Roman a n d JudeoChristian. Along with subject m a t t e r we were also t a u g h t wisdom: the wisdom of humility, the wisdom of listening w i t h respect and tolerance, the wisdom of never compromising our principles, the wisdom of logical thinking, a n d the wisdom of recognizing our continuous n e e d for acquiring more knowledge, for we r e m a i n always so very ignorant. At F o r d h a m Prep. I discovered extra-curricular activities. I joined the school newspaper, the debating society, the chemistry club, and the G e r m a n club. I was fascinated by interschool athletics, and so I applied for, a n d was selected on the t h r e e - m a n t e a m t h a t brought to the school two years in a row championships in Latin sight translation. For some mysterious reason, these victories did not get one-tenth the coverage in the school paper t h a t did any routine victory on the football field. Nor were there any trophies to take home. Seeing my great inclination toward science, to the astonishm e n t of the Principal, my father enrolled me into the Latinscience p r o g r a m rather t h a n the Latin~:]reek curriculum. I regret this decision. This deprived me of reading in the original the Iliad which my father could recite by heart. A n d all the science t h a t I learned in High School was quickly superceded by the elementary college courses. My favorite teacher at the Prep was our senior year E n g l i s h - L a t i n teacher, a young Jesuit scholastic, Mr. James Reid, S. J. He was a t r u e scholar. He told us on the very first day that he expected us to become scholars a n d to take first place in the English a n d Latin inter-Jesuit school competitions. The final exams in these schools were p r e p a r e d centrally and not by each school. Year i n - y e a r out, first place was t a k e n by the same school, where the students were specially picked for excellence. Mr. Reid's contagious e n t h u s i a s m for scholarship caught on, a n d at the end of the year we obliged him, as our class took first place in b o t h subjects. In his classes,

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discipline was never a problem. He kept us hypnotized by the knowledge a n d wisdom t h a t he was i m p a r t i n g on us. Some of us just went ahead a n d read in Latin the u n a s s i g n e d p a r t s of Cicero, for shear enjoyment. J u m p i n g ahead, I should like to tell about two other Jesuits, Fr. Cronin a n d Fr. Murphy, each remarkable in his own way. These were my professors of philosophy in F o r d h a m College. In those days, in Jesuit colleges, h a l f of the time in junior a n d senior years was devoted to philosophy - scholastic philosophy, 2 h every day. Fr. Cronin t a u g h t in junior year: logic, ontology, cosmology, a n d epistemology. He was an eccentric old man. He practiced yoga, and coached tennis. His first words to the class were: "Babies! (he always called us babies; as we realized, he was referring to our general ignorance) I will teach you real philosophy. I don't give a "dam" (and r e m e m b e r t h a t a "dam" is a little I n d i a n coin) about the pedantic stuff in your text books. However, on w r i t t e n exams, you m u s t give the official line:' So m u c h for scholastic philosophy! We learned about Leibniz a n d Descartes (he was a great a d m i r e r of Descartes), Kant, Hegel, a n d others. These were all listed in the official text books, but only as erroneous teachings to be refuted. After a while, he selected some students a n d assigned to each a p a r t i c u l a r t h i n k e r about w h o m he was to write a paper a n d present it in front of the class. My a s s i g n m e n t was B u d d h a a n d I was the first one to perform. For two m o n t h s I read books about B u d d h a a n d wrote my paper. On the assigned day, I a p p e a r e d with a long t y p e d manuscript. I faced the class and placed the paper in front of me ready to read it freely. As the bell rang, Fr. Cronin rose up from his seat, snatched the paper away from me, a n d said: "Professor, the next hour is yours:' I almost collapsed. But, here my Jesuit education saved me. I s t a r t e d speaking, a n d the lecture went well. Every few m i n u t e s he would i n t e r r u p t me w i t h questions, requests for clarifications, etc. At the end of the h o u r he walked up to me a n d said: "Now, you know w h a t we face everyday. Professor you did well:' This was my first experience in teaching a n d I h a d passed the test.

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His favorite subject was cosmology. He rejected with disdain the official, scholastic t h e o r y of Aristotelian prime matter, a n d substantial form. He glorified Boscovich's d y n a m i s m a n d was fully convinced t h a t Einstein's equation was the answer. I r e m e m b e r how, the day after the first atom bomb was exploded over Hiroshima, he d a n c e d a jig before the class exclaiming: "Babies I k n e w all along that Boscovich was right!" Father M u r p h y was just the opposite. He was the epitome of an old Jesuit: dry, always alert and composed, logical to the last detail, a n d absolutely clear a n d systematic in his presentation. He t a u g h t us psychology, strictly scholastic. He was a disciplinarian, but fair to the students. From the b e g i n n i n g he a n n o u n c e d his rule: should anybody be absent from a lecture, he had to give the reason before the next class. (In those days, there were no nonexcused absences.) At one point in the middle of the year, the chemistry majors had a very difficult exam in the afternoon. So we skipped the m o r n i n g class, his class. The next morning, when we came up to see him, he answered t h a t this was not a valid reason for absence. He added that we were unreliable, assuring us t h a t he would grade our exams fairly, but t h a t we should not expect any leniency from him. After this, it is not a surprise t h a t the six chemistry majors came topping the class in his course. As we sat down, he looked up at the class a n d said: "Mr. T i m a s h e f f leave the class:' Flabbergasted, I went out of the door. I waited outside. At the end of the hour, he came up to me a n d said in a calm voice: "You were absent yesterday. You know my rule; why did you not come up a n d give your reason?" W h e n I answered t h a t I had been a m o n g the b u n c h of chemists, he said: "I did not notice you:' Next day, as class started, he asked me to rise a n d addressed the class: "I t r e a t e d Mr. Timasheff unfairly yesterday. I apologize for t h i s : Such was his integrity! A l t h o u g h we noticed that, at times, he was doubling over in the middle of a lecture, he lectured to the end of the year, not missing a single hour. He gave the final exam, graded it and, after t u r n i n g in the grades, entered a hospital.

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Shortly thereafter he died of cancer. In those days, cancer was a totally incurable disease. It was obvious t h a t frequently Fr. M u r p h y h a d been in severe pain, b u t his sense of duty a n d responsibility a n d absolute self-discipline did not p e r m i t h i m to skip a single class. This is w h a t he was teaching us by i m p o s i n g the strict rules on class attendance. As he remarked, anyone's absence deprived the entire class of questions or c o m m e n t s t h a t he might make, a n d he greatly encouraged such participation.

From Engineering to Physics to Chemistry The spring of 1943 saw me graduate from the Prep a n d the start of a series of mishaps a n d chance events by which Fate guided me to physical biochemistry. I h a d always w a n t e d to be a civil engineer: to build dams a n d bridges. Therefore, I was intent on going to a college w i t h a p r e e n g i n e e r i n g program. I was told by my parents that, if I w a n t e d to go to college, I h a d to win a full scholarship: 55-year-old refugee professors h a d to accept whatever was offered a n d it was less t h a n generous. We lived in great modesty in the Bronx, a n d I h a d to live at home; there could be no t h o u g h t of going elsewhere to study. A n d so, together with t h r e e classmates I s t a r t e d t a k i n g the required exams for a New York State scholarship. O u r devoted teachers stayed after school to s h a r p e n us in each subject. All four of us were well on the way to qualifying, as we approached the last exam in the series. On the scheduled Saturday m o r n i n g we came to the school only to discover t h a t the principal h a d gone away overnight carrying in his pocket the key to the locked box with the exam questions. Automatically all four of us were disqualified. At Fordham, I h a d faculty tuition remission. Therefore, I s t a r t e d F o r d h a m College as a physics major, the next best p r e p a r a t i o n for civil engineering. At night a n d on weekends I worked as an usher at the Radio City Music Hall, a very interesting education in practical sociology. This was wartime,

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and, after my sophomore year, Fordham discontinued its physics program (most of the faculty had been drafted). All six physics majors decided to change over to chemistry. There was one problem: we had to catch up one year of organic chemistry. A young professor, Dr. Douglas Hennessy, volunteered to teach an extra session, and so we became chemistry majors. Dr. Hennessy was a superb lecturer. He could magnetize the class with the presentation of a synthetic pathway or raise suspense in the development of the proof of structure of a terpene or a steroid. (We must recall that in 1945, organic chemistry consisted of classes of compounds, and their reactions, proofs of structure of natural products, and synthetic pathways. The science of mechanism was only starting, and did not trickle down to undergraduate courses.) By the end of the year I was convinced that I wanted to be an organic chemist. When, years later, I joined Brandeis and signed up with a local young dentist, we discovered in conversation the amazing coincidence that Dr. Hennessy was his uncle. So, I heard much about this remarkable man. W h e n he retired from Fordham to Prince Edward Island, he set up a small laboratory where students would come up to do research. He also developed a deep interest in opera, and quickly acquired detailed knowledge of the history, scores, settings, and plots of many operas. The last achievement must e a r n the respect of many an opera lover who never could extricate himself from the "logic" of the action taking place in front of him. Dr. Hennessy lived to be past 90, leading an active life to the end. Senior year included a course in physical chemistry. In those days, physical chemistry was a totally new subject and not taught in many undergraduate programs. The man teaching the course was Professor George Antonoff. He did not know physical chemistry as we understand it. He had done his studies in St. Petersburg with Konovalov and Mendeleyeff. And so, we learned from him all about the ideal gas law, deviations from it, and gas phase compositions. He taught us how to do properly least squaring from experimental points using a slide rule

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(there were no computers or pocket calculators). We h e a r d all about the Antonoff Law of interfacial tension (an i m p o r t a n t contribution). We h a d an exhaustive analysis of first-order kinetics a n d why they depended on the n u m b e r of molecules a n d not the concentration, as chemists expressed it: he was a nuclear chemist a n d had discovered one of the isotopes of r a d i u m w h e n he was in Rutherford's laboratory. Over this he h a d a long dispute w i t h Marie Curie, a n d we learned all the details of that. But he did not know free energy a n d w h a t goes with it. So, I did not learn m u c h useful physical chemistry but he would tell me in his office about the scientists of the early 20th century, w h o m he k n e w well, s t a r t i n g from A r r h e n i u s a n d going down the alphabet. I have never forgotten his amazing story about being in Rutherford's laboratory as a Fellow of the Russian Empire (the title may not be exact). He was a p p o i n t e d d u r i n g the First World War a n d left St. Petersburg for Britain. On the way he decided to visit a good friend (a promin e n t scientist) in Denmark. As he told me: "X was either all-right or in one of his sessions:' He r a n g the doorbell a n d was greeted by a m a n with a glass in his hand. "So:' Antonoff said: "I joined him:' After three days he h a d missed the boat, a n d took the next one. W h e n he arrived in Cambridge, a friend who saw him, r e a r e d back a n d exclaimed in stupefaction: '~ntonoff, w h a t are you doing here? You are dead! This is a ghost!" As it t u r n e d out, the first boat h a d been s u n k by a G e r m a n submarine, a n d his n a m e was published on the list of victims. He concluded the story: "The m a n who s a n k was not I, it was an imposter Antonoff!" Because of the war, his Fellowship was extended indefinitely, and so he became, as he p u t it, the only Russian Imperial Fellow paid for by Lenin. The Soviet government, not w a n t i n g to offend Britain, kept up payments of all previous engagements until, after a few years, they uncovered the error a n d cancelled the stipend. In the middle 1930s he was h i r e d at F o r d h a m by a D e p a r t m e n t C h a i r m a n to w h o m nuclear decay seemed to be good physical chemistry. As I learned m u c h later, he retired at the compulsory

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age. There was no pension plan. He lived in great need a n d died in dire poverty.

Graduate School: F. F. Nord Having made the decision to become an organic chemist, on g r a d u a t i o n in 1946 I just stayed in the same building where I acquired a cubicle of my own for the next five years. I went to work for Professor F. F. Nord, r e n o w n e d as editor of Advances in Enzymology a n d founder of the Archives of Biochemistry a n d Biophysics. Dr. Nord was Professor of Organic Chemistry a n d Enzymology. I w a n t e d to do synthetic organic chemistry. Dr. Nord, however, looked at me straight in the face a n d said with his inimitable G e r m a n accent: "I know t h a t aggregation is i m p o r t a n t for enzyme activity. Study the problem. I want the first paper on my desk a year from t o d a y ' One year from t h a t day, he called me into his office a n d scolded me for my laziness, because there was no m a n u s c r i p t on his desk. He did not ever give any indication of how the problem was to be studied. My w e a k protests were dismissed with the remark: "I am giving you a piece of bread for your life:'And, in fact, he was right. He also gave me the following injunction: "You are now e n t e r i n g a monastic life in which work in the laboratory is everything:' He did call his students at m i d n i g h t or at 6 a.m. to point out a paper they should read a n d on one occasion, facing the anger of a w o m a n over being disturbed at such hours, with the remark: "I am his wife,' his reply was: "I am his mentor:' W h a t is more i m p o r t a n t ? A well-remembered incident o c c u r r e d one Saturday afternoon. L o o k i n g out the front door of the chemistry building, Dr. Nord saw one of his students, in the c o m p a n y of his fiancee, on the way to the football field. He hailed t h e m in a n d harangued the poor girl with: "Madam, you are dissipating this man. If he absents himself from the laboratory, he will never become a scientist, etc:' I do not know if they got to the football game t h a t afternoon. They did get m a r r i e d after he received his

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PhD. Dr. Nord held his students in terror. His b o o m i n g voice calling out someone's n a m e carried t h r o u g h the whole building. He expected us to be in the laboratory day a n d night. Yet, w h e n one of his students became seriously sick, Dr. Nord saw to it personally t h a t he h a d the best possible medical care. On the day after I defended my thesis, I walked into the c h e m i s t r y building, sort of lost. It was all over and I did not know w h a t to do. Dr. Nord saw me a n d beckoned me into his office. I entered in awe as always. He t h e n offered me a chair a n d in a calm friendly voice said: "Dr. Timasheff, sit down, Herr Kollege:' It is t h e n that I u n d e r s t o o d t h a t his a u t h o r i t a r i a n m a n n e r s were his way of keeping us at the bench. He told me one day "My rough m a n n e r s only reflect t h a t I was a sergeant in the P r u s s i a n army:' A n d indeed he was a G e r m a n from Prussia, who e m i g r a t e d w h e n Hitler came to power. He was a believer in democracy a n d was a m a n neither of the right nor of t h e left. A m o n g the professors t h a t I k n e w at Fordham, Dr. Victor Hess, who h a d won the Nobel Prize for his discovery of the cosmic rays, was an Austrian Catholic; Professor Einaudi, whose father was elected as first president of Italy after the war, was an Italian Catholic; Professor Baerwald, who was responsible for b r i n g i n g my father to Fordham, was a G e r m a n Catholic. All h a d left for the same reason as Dr. Nord. None of their lives h a d been threatened. How in 1946 did one study as precise a problem as w h a t h a d been assigned to me? In fact, a start h a d been made. One year earlier t h e same problem h a d been assigned to a n o t h e r graduate student, Milan Bier, a n d I was to follow him. The study of aggregation requires m e a s u r e m e n t of molecular weight. How to measure it? The one piece of "major equipment" in Dr. Nord's laboratory (and in the whole department) was a B e c k m a n DU spectrophotometer; a year later a pH meter was added; we cast our dialysis bags from collodion. It was 1946 a n d Debye h a d just invented the m e t h o d of light scattering for m e a s u r i n g the molecular weight of high polymers. N o t h i n g had been published, but Milan h e a r d Debye give a s e m i n a r at Columbia University,

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where the great man described the method as essentially shining a flashlight at a test tube with the solution and looking at it at a right angle. What you saw gave the molecular weight. W h e n I joined the project, Milan had started the construction and I was to develop the collimating system in the optics. We had a total budget of $50 and no mechanical shop. Visits to pawn shops, scavenging galvanometers discarded by the physics laboratory, and all sorts of improvisations led to the construction of a working light-scattering instrument which permitted the two of us to obtain our PhDs. In doing this, we had to improvise and use our imagination. A collimating system requires a pinpoint hole in a rigid disk. I discovered that the diaphragm from the public phone in the lobby was exactly what was needed.

Solving the Wrong Problem The problem which we studied t u r n e d out to be not enzyme aggregation, although both of us did do some work with egg albumin, one of the half-dozen proteins that one knew how to isolate. The problem fell on us by accident. W h e n the lightscattering instrument was built, it needed to be tested. Now, Dr. Nord happened to own a battery of jars labeled polyvinyl alcohol (PVA) with different batch numbers. This was to be the medium for testing the stability of our instrument. From the beginning we noticed a drift in the intensity of light scattered which fluctuated slowly back and forth with time. Weeks went by, as we perfected at infinitum our optics, looking for reflections, and our technique for removing the dust which is the proverbial nemesis in light scattering, until Milan noticed that the fluctuations had a regular periodicity over day and night. It was winter and what varied was heating of the room. Controlled heating and cooling of the polymer solution revealed that its turbidity went up at higher temperature, reversibly, i.e., PVA aggregated reversibly on heating. We had fallen

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obviously upon a problem in polymer solution chemistry. How to analyze that? Now, as I have mentioned already, in 1946 Fordham had only a rudimentary instruction in physical chemistry, my understanding of free energy was sketchy and chemical potential was essentially a mystery. To overcome our ignorance in this field, Milan and I started attending the Saturday morning polymet symposia r u n by Herman Mark at Brooklyn Polytech. Following a one-and-half hour subway ride, we would hear such luminaries as Lars Onsager, Raymond Fuoss, Aharon and Ephraim Katchalski, Victor La Mer, J. J. Hermans, and others. At that time, Flory and Huggins were developing their theories of polymer solutions. A transformation of the light-scattering equation to their theoretical equations gave a thermodynamic interpretation of our observations and led to our publishing the first application of their theory [6]. It t u r n e d out that reversible aggregation on heating was increased with an increase in the degree of acetylation of PVA. We attributed the aggregation to a hydrophobic effect, only to be told by a journal referee to remove that "meaningless remark:'

Kirkwood: Caltech, Yale Then came the day for which each one of us had longed. Dr. Nord called me into his office and in a solemn voice pronounced: "When you go to the American Chemical Society meeting to give your talk, you should register at the Employment Clearing House. When you return, you must prepare your dissertation:' This gave me exactly six weeks to get my dissertation in time for the deadline: it was done. In that, Dr. Nord's requirement that each month every student had to present a detailed written report was of great help. I went off to the meeting, fully expecting to get a job in some northern New Jersey chemical industry. When, at the end of the first day, I picked up my interview slips, there were half a dozen appointments from industry and also one signed John G. Kirkwood, Caltech. I could not believe

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what I saw; this was beyond the dream of anyone at Fordham. On seeing this slip, Dr. Nord said: "Kirkwood! I would go to work for him if I had to pay him" Now Kirkwood had, as a relaxation, a small experimental project, on the level of two postdoctorals. John Cann had just left and Kirkwood was looking for a replacement. With trepidation I went to the interview, where I was faced by a kind, somewhat shy man. After 20 min, he offered me a postdoctoral fellowship. And so, in February 1951, off I went to Caltech, and, after six months, I moved to Yale with Kirkwood for an apprenticeship that lasted four years. The project was one of protein fractionation by the method of electrophoresis-convection, which Kirkwood had invented from first principles. As I recall the story, Kirkwood, who was working on some aspect of the M a n h a t t a n project, was sitting in a train. He always had an interest in proteins. In thinking about the diffusion process for purifying uranium, he got the idea that the same could be done for proteins. What was needed was a cell with a long narrow channel equipped with electrodes on both sides. Proteins carried by electrophoresis would accumulate against one wall (a dialysis membrane) of the channel and the density gradient would result in a laminar flow until only the protein isoelectric at the given pH would remain in the top compartment. He derived the equations, then hired a postdoctoral to construct the appropriate apparatus. The method worked and the equations described the results exactly [7]! At Caltech I found myself immersed into a maelstrom of science. On my first day, I saw Pauling unveil the s-helix (along with some other structures which turned out to be wrong including the inside-out DNA double helix). I was carried along by a continuous discussion on questions of protein homogeneity, self-association, immunochemistry, and solution thermodyamics. Harvey Itano and Jon Singer had just demonstrated by Tiselius electrophoresis that sickle cell hemoglobin differed from normal hemoglobin by one charge, the first discovery of genetic variants in proteins. I realized that I was totally ignorant (the plight of

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many who arrived at Caltech after being star students at various institutions). The answer was: study, listen, and learn. In 1950, Kirkwood together with his graduate student, Dick Goldberg, published the theory of light scattering, which developed multicomponent interactions [8]. When, on arrival at Caltech, I met Dick Goldberg, his first words were: "You know, light scattering does not give just the molecular weight, but there are interaction terms:' I had practiced light scattering, while Dick had never seen an instrument. This comment caught my attention and eventually opened the road to a major part of my research experience. Kirkwood looked upon his experimental group as a relaxation, a hobby. At the end of the day, he would frequently walk into the laboratory with the remark: "How are things going?" He would then listen with amusement to the description of some experimental difficulties and give a few words of encouragement. I remember when I told him one time that a fractionation was not working because the dialysis membrane was getting clogged. He thought a minute and, after two puffs on his pipe, answered: "What you are trying to tell me is that, in this case, the expansion in the transport equation does not converge:' Each week I had my standing half-hour with Kirkwood. He would frequently make some r e m a r k about solution thermodynamics, or some protein physico-chemical properties, and then would ask me "Do you want me to explain this to you?" I then listened avidly and took notes. And so, over four years of private tutoring from JGK, I learned all that I could about solution thermodynamics, protein electrostatics, hydrophobicity, and hydration, among other topics. E F. Nord had taught me how to do research; Kirkwood taught me the science that I practice. The years at Yale were very fruitful. Kirkwood organized a protein discussion group with the participation of various faculty members, postdoctorals, and graduate students. The range of topics was very broad and the discussion animated. At one point, I recall the discussion t u r n e d to the origin of life. A very popular theory had just been proposed by Ovchinnikov

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that simple organic molecules were first generated by highenergy lightning striking the proper environment, with more complex structures gradually developing, eventually leading to life. W h e n a consensus was established that the probability of this happening was essentially nil, one faculty member exclaimed: "If not this, then what? It had to happen somehow:' Kirkwood looked up and remarked in a low voice: "There is a much older, but simpler theory:.' He did not elaborate, nor did the topic come up again. I continued to work with electrophoresisconvection. In 1952 Kirkwood with his student, John Shumaker, published the theory of charge fluctuations on proteins [9]. It stated essentially that the fluctuation of charges and charge configuration on a protein surface should result in an attractive force between the protein molecules. This predicted that the light scattering of an isoionic protein should follow a dependence on the square root of protein concentration. So, back I went to light scattering. In this, I was soon joined by Howard Dintzis who arrived for a postdoctoral. Howard, besides having a highly critical intellect, was a magician in the laboratory. He realized that the technique we were using was too cumbersome. He quickly developed completely novel techniques of preparing protein solutions and doing rapidly multiple scattering measurements. This increased our productivity ten-fold. Hundreds of points were measured and the K i r k w o o d - S h u m a k e r theory was proved experimentally [10]. Kirkwood died from cancer in 1959 at age 59. Among other things, he was a prominent expert on underwater explosions. This caused him to be an observer from the bridge of a warship of the underwater nuclear explosion in the Pacific. He made the r e m a r k once that he had been sprayed and that he believed this was the cause of his cancer. At the time the dangers of radioactivity were poorly understood. Several other scientists who had participated in these essays also died early. In my second year at Yale, I met M a r i n a Gorbunoff, a graduate student in organic chemistry. We got married and, in later years at Brandeis, she became a key member of my research

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group. M a r i n a Gorbunoff belongs to the Second Russian Emigration, i.e., to the two million who decided to leave Stalin's paradise d u r i n g the G e r m a n occupation of large p a r t s of the Soviet Union. As my mother-in-law said: "I did not know where I was heading, but I did k n o w one thing: I was r u n n i n g away from Stalin's hell:' A n d so, w h e n the p a r t of the Caucasus where she was with her child was overrun by Germans, she set out west. After a two-year saga t h r o u g h the Ukraine, Poland, a n d Latvia, which included evasions from Soviet partisans, as well as the Gestapo, they ended up in Germany, in M a r b u r g an der Lahn. Similar stories can be told by t h o u s a n d s of others. W h e n the war ended, M a r b u r g t u r n e d out to be in the A m e r i c a n zone. This was fortunate. In Yalta, the w e s t e r n allies h a d signed a n a g r e e m e n t t h a t they would repatriate to the Soviet Union all nationals of t h a t country, both prisoners of war and civilians. Soon, Soviet repatriation commissions a p p e a r e d on the scene a n d p r o t e s t i n g people were h a n d e d over to them. There were massive suicides. Many of the prisoners of war went back w i t h joy. The destination for all was the Gulag, if not the firing squad. W h e n m u c h later it became possible to correspond with relatives in the USSR, my wife learned t h a t her uncle, who had been a prisoner of war, r e t u r n e d home only after t e n years spent in the Gulag; he was totally lame a n d blind. As this situation became known, General Eisenhower made a h u m a n e decision: in order to repatriate anyone, the Soviet authorities h a d to produce proof t h a t the individual belonged to them. This essentially ended repatriation from the A m e r i c a n zone. The British c o n t i n u e d the practice and, in their zeal, some t u r n e d over ~migr~s from the 1920s who h a d been deprived of citizenship by t h e Soviet Union a n d m a n y of w h o m between the wars h a d settled in west E u r o p e a n countries. M a r i n a Gorbunoff s t a r t e d studying chemistry at M a r b u r g University u n d e r the great Meerwein a n d in 1950 she, together w i t h her mother, i m m i g r a t e d to the U n i t e d States u n d e r the Displaced Persons Act. Two years later she was accepted at Yale Graduate School. Marina's m o t h e r was a Terek (Caucasus)

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cossack. In the 19th century t h e Cossack villages were raided periodically by the Chechen, who abducted young m e n to work as slaves. In one such raid, Marina's great grandfather was abducted. After 30 years he was released, as he was no longer useful. W h e n he rejoined his wife, they adopted two U k r a i n i a n orphans, one of w h o m was Marina's grandfather. This brings to m i n d Lermontov's lines (11): "Streams the Terek on the stones In splashing murky waves, A fierce Chechen crawls to its shore Sharpening his dagger:' Her father, who was of Mordovian descent, was an engineer. His specialty was the design and c o n s t r u c t i o n of chemical plants. D u r i n g the war, he h a d r e m a i n e d in Moscow. He was shot in Stalin's postwar purge. P u r g e s seem to have been a hobby with h i m who, d u r i n g t h e war, h a d been k n o w n as "good uncle Joe:' "beloved by his people" who presided over "another k i n d of democracyf.' His purges a m o u n t e d to a total of 50-80 million "loving subjects." The Second Emigration was, to a great extent, a replay of the first one. People w i t h higher education took whatever work they could find. I recall a man, who h a d been a Professor of chemistry at the University of Kiev, working in a hospital w a s h i n g chamber pots. They worked a n d slept in shifts, giving their children an education. Now, fully i n t e g r a t e d into A m e r i c a n society, a n d going into the third generation, they occupy all the various professions, e n g i n e e r i n g being a favorite one. With them, "Little Russia" saw a second life, but this time it centered on the preservation of one's cultural heritage, as no one was expecting to "return home:' A n d this heritage was preserved. I recall, on the occasion of a s e m i n a r trip, meeting in the mid-1980s a young scientist from the Soviet Union. After a few minutes of conversation, he exclaimed "You are our history!" A n d so it was. "Little Russia" kept t h e culture from which the

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Russian people had been cutoff for 80 years. "Little Russia" s p a n n e d the gap a n d now is helping the newly liberated people to find their roots. On more t h a n one occasion I have h e a r d the remark: "We are lost, we are t r y i n g to find our roots:' Sometimes in an awkward way, they are reaching out to the past. In the t h i n t h r e a d of the r e m n a n t s of "Little Russia" they at times see a bridge over the 80-year long cultural a n d historical void. Finally, I was ready to go on my own a n d apply w h a t I h a d learned. After negative answers from the two score universities where I h a d applied, I accepted one of the two offers which I had. It was from the E a s t e r n Regional Research Laboratory of the US D e p a r t m e n t of Agriculture in Philadelphia. I was disappointed: m a n y of my friends h a d obtained faculty positions in various universities. I h a d to be satisfied with a rather obscure institution. Yet, again Fate had directed me properly: the place t u r n e d out ideal for developing myself freely as an i n d e p e n d e n t scientist, in a totally sheltered environment. I left New Haven in J a n u a r y 1955 a n d went to Philadelphia. My wife, who received her PhD in J u n e 1956, at the commencem e n t which saw Konrad Adenauer receive a Yale h o n o r a r y degree, was continuing her studies. So for the next 16 m o n t h s I c o m m u t e d to New Haven every weekend. In those days it was a comfortable 4-h t r a i n ride. On Saturday mornings, I c o n t i n u e d to go to the chemistry building where I would visit Kirkwood. Late in this period, Charles Tanford came to Yale for a sabbatical, d u r i n g which he developed his classical t h e o r y of protein ionization w i t h a discrete distribution of charges [12]. We h a d k n o w n each other from the Eli Lilly insulin conferences: I was t r y i n g to fractionate insulin by electrophoresis-convection, while Tanford was working out its electrostatics. We s t r u c k a close friendship which has r e m a i n e d for life. I n the second h a l f of the fifties a n d early sixties we used to go to the Gordon Research Conferences on Proteins. At one of these conferences, Charles helped me to learn t h a t in science recognition is frequently given on the basis only of pedigree. One of the m a i n topics was hybridization between genetic variants of

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hemoglobin. We h a d done hybridization experiments with the genetic variants of fl-lactoglobulin, t h e only other p r o t e i n k n o w n at the t i m e to have genetic variants. As the hemoglobin story unfolded I realized t h a t t h e two systems behaved in different ways: the hemoglobins formed hybrids, fl-lactoglobulin did not hybridize [13]. In the discussion period, I raised my hand. The C h a i r m a n (John Edsall) did not recognize me. I kept my a r m raised, as others were recognized, until I t h o u g h t I would get cramps. Tanford p u t up his hand. He was immediately recognized. He said: "I believe t h a t Dr. Timasheff has some c o m m e n t s to offer:' I was recognized. As I gave my two slides to the projectionist a n d was r u s h i n g past the Chairman, he whispered to me: "Not more t h a n 30 seconds:' I showed the two slides a n d spoke for 3 min. As the session ended, Charles came up to me and said: "Serge, now you k n o w w h a t mistake you made by never going to Harvard:' Next day, I h a d a very fruitful discussion with the hemoglobin people. It was obvious t h a t hybridization was a m u c h more complex p h e n o m e n o n t h a n t h o u g h t until then. To this day my wife a n d I continue our tradition of m e e t i n g periodically w i t h Charles Tanford a n d the energetic, erudite, a n d so full of sense, Jackie Reynolds. She is one of the several examples t h a t I know of a w o m a n who p u r s u e d a full scientific career while raising a family. These days our get-togethers can be in L o n d o n or Paris, or some small t o w n in France w i t h a good restaurant. The last is an absolute requirement. On one such occasion, Charles was the cause of my learning t h a t pedigree a n d i m p o r t a n c e are keys to attention, not only in science, but also in the world of restaurants. It was m a n y years ago in New Orleans where I went for some scientific meeting. Together w i t h Charles a n d Jackie we went to one of the famous F r e n c h restaurants, w i t h menu-in-French. As we entered, Charles introduced me as a great connoisseur of food and wine from Paris (true, I was b o r n there a n d I did appreciate good cooking). I was s u r r o u n d e d by the Maitre d: the wine steward, a n d the table waiter. I decided to play out the i m p r o m p t u role; I spoke only F r e n c h a n d asked deep questions about the p r e p a r a t i o n of the

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various dishes. The chef came out. They, obviously, knew little French and were facing a critical situation. In any case, we enjoyed the meal immensely, the service was excellent, the chef exerted himself, and the wine steward was all attention. As we were leaving, they were all lined up at the door and, as we all shook hands, I made some complimentary remarks, speaking in my best French style English. They were all in smiles.

Working for the Government At ERRL I was assigned to the section led by Brooks Brice, a specialist in physical optics, who had developed the then standard instruments for measuring light scattering and differential refractometry. I had the position of Head of the Physical Chemistry Unit, which consisted of me and an associate, Bob Townend. In the choice of problems, I was restricted to one of the commodities assigned to the Eastern Region. I chose to work on fi-lactoglobulin which is a component of milk. Together with Bob Townend, who had great dexterity in the laboratory, we characterized fully the self-association properties of the genetic variants of fl-lactoglobulin. We did this by velocity sedimentation [14].The results were analyzed in terms of the theory that Geoffrey Gilbert had just developed [15], the first application of his theory. Then we verified the conclusions by light scattering [16]. This was in keeping with the practice which I adopted: studies must be done by two unrelated techniques with the requirement of quantitative agreement. This was the first such study on record. Briefly, fl-lactoglobulin (fl-lg) is a dimer of identical subunits. In the pH region between 3.5 and 5.2, it undergoes a reversible tetramerization reaction to terminal octamers. We noticed early that the results could be analyzed only if the protein was a mixture of two species, one which could self-associate and the other which could not. Aschaffenburg's discovery of two genetic variants of protein, fl-lg A and B [17], permitted us to identify A as the

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self-associating species, while B was close to inert [18]. Bob Townend then showed by peptide analysis that the two variants differed by two amino acid substitutions, Asp in A for Gly in B and Val in A for Ala in B [19]. It t u r n e d out that the aspartate residue in question was involved in the tetramerization, which explained the difference in behavior. Electrostatic analysis permitted to account fully for the pH dependence of the phenomenon. There is an interesting corollary to this story. The hemoglobin genetic variants all differed by a single amino acid substitution; fl-lg A and B, however, differed by two. I got interested: how could this happen? To my inquiries with several well-known geneticists, I was told categorically that each substitution must correspond to an independent mutagenic event. My suggestion that a hit with a high-energy particle of the DNA folded in a chromosome might affect two sites brought into juxtaposition by folding was universally dismissed with the r e m a r k that the genetic information is a r r a n g e d linearly along the DNA, rendering it impossible to hit two linearly distant sites simultaneously; in 1960, three-dimensional thinking was practically nonexistent. This set us on a search. In collaboration with the USDA animal husbandry scientists, who had access to miriads of cows all over the world and knew their genealogy, we screened the milk of close to one thousand cows. We never found a fi-lactoglobulin intermediate between A and B [20]. In this basic research at ERRL, I had the full support of Brooks Brice and Thomas Mac Meekin, one of the classical early protein chemists. In particular, I am indebted to Percy Wells, the Director of ERRL. He gave me absolute freedom to do what I wanted. I was given a Model E ultracentrifuge, a Tiselius-free electrophoresis apparatus and, of course, light scattering. Percy Wells was a most unusual USDA administrator. As he told me later, it was his policy to identify a few talented young scientists at ERRL and let them develop freely. I belonged to that category, as did Heino Susi, renowned for his in-depth development of the infrared spectroscopy of proteins. Heino and I shared many beliefs and principles. He was the son

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of the Estonian whom Solzhenitsyn met in the Gulag and who introduced that greatest fighter for freedom to the concepts of freedom and democracy. On his visits to Estonia, Heino smuggled out of the Soviet Union some of the Solzhenitsyn manuscripts. He died just before the liberation of Estonia for which he had worked many years. Another good friend who died before he could see the liberation of his country, Hungary, was George Nemethy. George was forced to escape from Hungary in 1956 when the Hungarian people who had liberated themselves were abandoned to Soviet tanks. George was a very gentle intellectual and a highly cultured man. Returning to ERRL, what made our free scientific development possible can be summarized in Percy Wells' remark: "No written project (always totally applied) has ever prevented us from doing good science:' When he retired, his successor imposed a diametrically opposite philosophy. It was in this spirit that Percy Wells, who knew how to bypass government red tape, happened by chance to play a major role in the launching of penicillin production during the World War II. One day, while waiting in the outer office of a Washington administrator, he found there two Englishmen. They happened to be Florey and Heatley who were being shuffled from one government agency to another. When, in casual conversation, he learned what they were seeking, he immediately identified their needs and directed them to the fermentation facility at the Northern Regional Laboratory. A few phone calls and production started. Without this chance encounter, the connection might have never been made. While at ERRL I also developed an appreciation for applied research, as I collaborated informally with the very competent engineers on their whole milk powder project. One of the delights in working on proteins in those years was that all protein chemists (and they were mostly physical chemists) knew each other and shared their latest findings. The messengers were frequently ultracentrifuge service engineers who traveled from one laboratory to another and told what they had learned on their various visits.

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My interests in protein interactions extended beyond selfassociation and simple ionization. One of the topics of general interest at the time was long-range order. I found particularly intriguing the conclusion reached in 1952 by Kirkwood and Mazur that ordering could occur in solutions of globular proteins under conditions of high protein net charge in a solvent of very low ionic strength [21]. At high enough protein concentration this could lead to gel formation from repulsive forces alone. This should manifest itself through an effectively very large excluded volume which would lead in light scattering to a very large nonideality term, of the same magnitude as the molecular weight of the protein. We did measurements with a-lactalbumin (a milk protein) and indeed observed the effect [22]. A much better approach would be small-angle X-ray scattering, where maxima and minima should be seen in the scattering envelope. The ideal place to do the work seemed to be Sadron's Centre de Recherches sur les Macromolecules (CRM), where Vittorio Luzzati had the necessary facilities. A great attraction was Strasbourg itself, a magnificent Alsatian city with its fabulous cathedral and lovely houses along the canals. This was surrounded by delightful villages, where one could enjoy delicious wines and a splendid cuisine. The proximity of the Vosges mountains, Basel, the Black Forest, Frieburg, and Baden-Baden added to the magnetic attraction. On a vacation trip through Strasbourg in 1958, I talked withVittorio Luzzati who expressed his great interest. Dr. Wells agreed to my going away on leave without pay. All that was needed was a stipend. Shortly before, NSF had started its program of Senior Fellowships. I applied and was awarded one. We were on our way to Strasbourg.

I am a USDA Bureaucrat in Rome

We left in early summer of 1959 and returned in January 1961. Our itinerary was not exactly straight. We took an Italian Line ship (the m/v Saturnia) to Lisbon and, after exploring the very

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interesting cathedrals of Portugal for three weeks, by train a n d bus (many areas of Portugal at that time still had no electricity), we arrived via Sevilla in Algeciras (next to Gibraltar). I can still feel the bright July Portuguese sun, relentless, in a deep-blue sky. I r e m e m b e r how I stood in the shade of a telephone pole waiting for a train, slowly shifting as the Sun moved in the sky a n d how, on arrival in the town of Evora, with its cathedral, the two of us c o n s u m e d w i t h i n a few m i n u t e s 2 L of water in our pension room. In Algeciras we boarded the S/S Julio Cesare a n d sailed via Barcelona a n d Genova to Naples. In Naples we got off the ship a n d traveled to Rome where, for one month, I worked for the US D e p a r t m e n t of Agriculture. After the war, the US Government a c c u m u l a t e d large sums of money in various countries as payment for diverse aid. It agreed to spend this money on the spot (P. L. 480). One of the ways it spent it was t h r o u g h research grants, a d m i n i s t e r e d by the D e p a r t m e n t of Agriculture. For Europe, the center was Rome. I asked Dr. Wells if I could stop there for one m o n t h to review grants. He told me that I h a d to get permission from Dr. Shaw, the Head of Agricultural Research Service. I went to Washington and presented myself in Dr. Shaw's suite. Now, access to Dr. Shaw was not easy. I was announced, and, after a few minutes, the door opened and I entered. In the office I noticed some Yale memorabilia. I found Dr. Shaw to be a very affable man. We discussed what I could do in Rome. In fact, grant reviewing was progressing slowly a n d they could use help. He asked me about my Yale experience. W h e n he learned that my wife was a Yale PhD his smile broadened even more. He approved my a p p o i n t m e n t in Rome for one month. I had discovered the m e a n i n g of the Yale connection. It had opened the door of the Director's office. W h e n I got to Rome, I found that the scientific director also hailed from Yale. In their home, we sat on Yale chairs and dined from Yale china. We were t r e a t e d as members of the family. I spent my time reviewing grants, while my wife visited the sights and museums. Frequently, I ate lunch in a delightful trattoria with the Director. In Rome I also met an amazing

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business administrator. With him, we ate lunch at the A m e r i c a n bar in a large hotel onViaVeneto. One time, I tried to talk about antiquities. His c o m m e n t was: "Once you have seen one old stone, you have seen t h e m all" He gave me invaluable lessons on how to deal w i t h the bureaucracy: first, never to forget that, for a rule that says NO, there is another one that says YES; second, to use proper wording. (The first was brought to m i n d by a r e m a r k t h a t I heard m u c h later from an Assistant Secretary of Agriculture for Administration, as he addressed a general audience; he t u r n e d to a high-ranking administrator with the words: "Get out of channels, Paul. Remember, rules are made but to be broken!") The second rule was checked when, m u c h later, I almost caused a crisis by offering a s e m i n a r speaker an honorarium. The whole t h i n g calmed down when I c h a n g e d it to a lecture fee. After this, I never e n c o u n t e r e d any difficulties with administrators. They all expressed their appreciation of a collaborating scientist (a rare phenomenon).

Strasbourg, Sadron, Small-Angle X-ray Scattering In early September 1959 I arrived at Sadron's Centre de Recherches sur les Macromolecules (CRM). Charles Sadron was famous for his development of the concerted hydrodynamic analysis of polymer folding in solution. He was an idealist. His life was m a r k e d by many tragedies, not the least of which was the death of his 20-year-old daughter from the same debilitating disease that h a d carried away her mother. D u r i n g the war he h a d been deported to Dachau. Yet, after the liberation, he crossed the Rhine to extend his friendship to his G e r m a n colleagues. This was one of the first steps in the reconciliation between the two countries. We saw h i m with his second wife (a c h a r m i n g French lady) many times over the years, first in Strasbourg, t h e n in Orleans when he opened a new research institute there. Over the years I collaborated with several members of his research establishment. Most memorable was

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his visit to Brandeis, when he was finally g r a n t e d a visa to the USA (at one point he had signed a "wrong" declaration). He gave a stirring s e m i n a r on the physical s t r u c t u r e of DNA, a n d entert a i n e d students with tales of his travels to China. Our arrival in Strasbourg was rapidly followed by that of Giorgio a n d Gabriella Bernardi, who were to become our lifelong very close friends. We first got to know each other in our frustration over looking for an apartment, a convoluted procedure those days in Strasbourg in which one was approached by all sorts of bizarre intermediaries who "knew" of an a p a r t m e n t and could lead you to it. Finally, we got settled. Our rental ended with a lawyer. The story is again one of coincidence a n d worth telling. As we were coming to the end of our stay, our landlord, w h o m we had never met, presented us with a bill for a large sum of money (some hidden clauses in the w h a t t u r n e d out the illegal p a r t of the contract). Confused, I addressed myself to Sadron's secretary for advice. She just said: "Give a call to Maitre B, he is a good friend of Professor Sadron:' I did. I knocked on the door a n d was told to come in. I entered a modest office and found facing me a k i n d m a n in his sixties. He looked at the documents and asked me to refer the landlord to him. W h e n I m e n t i o n e d Maitre B to the landlord's Strasbourg representative, he j u m p e d up from his chair. Three weeks later, the m a t t e r h a d been settled, we did not have to pay the money. W h e n I told the story to an old Strasbourg resident, I got the resolution of the puzzle: the landlord belonged to a family of wartime collaborators who had accumulated a fortune by realestate speculation d u r i n g G e r m a n occupation. Maitre B was, in fact, a highly r e n o w n e d trial lawyeI: He is the one who h a d put members of that family in jail. Such is fate. Finally, w h e n I went to Maitre B to ask for his bill, he simply said: "You owe me nothing: we must protect foreign visitors from local crooks:' In Giorgio Bernardi I found a splendidly educated man, not only in physics a n d the biological sciences, but also in literature a n d art. He h a d an expert knowledge of the Italian Renaissance.

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We rapidly became close friends. Giorgio is an example of a superb young Italian scientist who became expatriate because of lack of opportunity in his own country. (At the present time, this is the t u r n of many brilliant young French scientists, who move to w h a t is referred to as the anglo-saxon countries.) Scientifically at CRM, we found a claustrophobic atmosphere. To liven things up, at our joint initiative, a p r o g r a m of internal research seminars was started. Giorgio gave a few talks on his work. I t a l k e d about protein interactions in solution, thermodynamic mechanisms, electrostatics, etc. There were hardly any comments from the polite audience. No one else volunteered to talk. The brilliant Henri Benoit s u m m a r i z e d this for me: "Cher Ami, France has never h a d an inclination for t h e r m o d y n a m i c s " This I found to be mostly true. (One should be r e m i n d e d that the t h e r m o d y n a m i c s of allosterism were developed by Wyman. As Wyman told me, Monod was totally u n i n t e r e s t e d in this approach; it was Changeux who recognized the power of the Wyman analysis.) While the m a i n t h r u s t of CRM was on the gross s t r u c t u r e of DNA, Giorgio realized the i m p o r t a n c e of individual segments for genetic control. He discovered "acid DNase" which cleaved at a p a r t i c u l a r base sequence [23]. In fact, it was the first finding of a restriction enzyme. Over the years, we collaborated in some conformational studies. At CRM, Bernardi's work did not generate enthusiasm, a n d so he h a d little support. I i n t r o d u c e d h i m to the U.S.D.A's P.L. 480 program in Rome and he applied. I helped by steering h i m t h r o u g h the cumbersome p r o c e d u r e a n d p u s h i n g the reviewing in Rome. He was f u n d e d a n d could pursue his research. This to me fully justified my earlier stay in Rome: my experience in bureaucracy h a d helped to support good science. Giorgio soon moved to Monod's institute in Paris, a n d we met frequently there or in various delightful places in Italy. His c o n t i n u e d involvement with DNA molecular genetics required fish t h a t lived in hot water. The tales of his travels to various parts of the world, some outright dangerous, to get the fish are fascinating. It is these tales t h a t inspired us to visit the Galapagos Islands

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(where I was assaulted by a booby bird), the Beagle channel, and Lago Argentino, sites of incomparable beauty. He is now the President of the prestigious Anton Dohrn Zoological Station in Naples, his birthplace, into which he has injected new life with his limitless energy. We were invited to the inaugural symposium. The talks have opened to me a whole new world, that of molecular evolution. At CRM, I did small-angle X-ray scattering with the effervescent Vittorio Luzzati and his imperturbable student, Jean Witz. The long-range order problem was giving difficulties and so we switched to another problem. One of the "hot" questions was the solution structures of DNA and RNA. DNA was readily available. RNA was hard to come by; in fact, it was, at the time a poorly identified substance. It so happened that my good friend, Ray Brown, whom I met at Caltech when he was Kirkwood's other experimental postdoc, was working at Lederle on the solution physico-chemical characterization of ascites tumor cell high molecular weight RNA, which had been isolated by John Colter [24]. Light scattering seemed an ideal method for measuring its molecular weight and overall shape. We collaborated and had just finished a paper on the work. When Vittorio heard of this, he became all excited and urged me to get a sample: small-angle X-ray scattering could give details on folding of the chain. A phone call to Pearl River and a frozen packet arrived in Strasbourg. Jean Witz and I did the experiments and Vittorio the final analyses. The conclusion was: RNA consists mostly of short double-stranded regions joined by short flexible regions [25]. This was the first demonstration of this structural fact, and was contrary to the then dogma that RNA was a single-stranded random coil. A concerted analysis of light scattering and small-angle X-ray scattering results led to a general overall picture of the molecules [26]. While there, I also induced Luzzati to look at the structure in solution of the fl-lactoglobulin octamer. Together with Jean Witz we showed it to be a compact "tilted cube" with 422 symmetry, identical with the unit cell in X-ray diffraction [27]. This was the

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first time that the structure of a protein oligamer had been seen in solution.

B a c k to Philadelphia: Solvent Interactions At the time of my stay in Strasbourg, Luzzati had a major program on DNA. His observation that the measured mass/length ratio in concentrated salt solution was too small for a double helix led me to realize that X-ray scattering, being a fluctuation phenomenon, had to have the same multicomponent factors as light scattering. I extended the Kirkwood-Goldberg theory to small-angle X-ray scattering [28] and deduced the excess of water on the double helix. At that point, my interest t u r n e d to protein-solvent interactions in general. The method used at first was light scattering. Later, following the development in Gratz of the high-precision densimeter, this became the technique of choice. In early papers, Milton Noelken, a postdoctoral from Tanford's laboratory, measured the preferential binding of guanidine hydrochloride to bovine serum albumin [29]. Hideo Inoue, my first postdoctoral from Japan, showed that the helixinducing denaturant, 2-chloroethanol, was preferentially bound at lower concentrations and excluded at high levels [30]. Inoue then showed how the surface interaction with solvent components could be formally decomposed into the effective numbers of water and other solvent molecules in contact with the protein [31]. Eugene Pittz who, while a student of Jake Bello, had been involved in the crystallization of RNase A from a water MPD (2-methyl-2,4-pentanediol) medium, measured the very strong preferential hydration of RNase in the presence of MPD [32]. This explained why that cosolvent is a good crystallizing agent. Looking over the postdoctorals who did solvent interaction work over the years, all, except for Milton Noelken and Eugene Pittz, were Asians. They were Chinese, Japanese, or Indian. Others had tried and were not successful. I have given thought

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to this observation. These studies are very slow. They require m u c h patience and extreme meticulousness in the experiments. This m e a n s continuous attention to details. The Asians t h a t I have k n o w n have these character traits. These days Western students, with few exceptions, want results fast and publication rapid. This is reflected by less careful laboratory habits, impatience over spending one day at the bench for obtaining one single point on a curve. My last postdoctoral, Guifu Xie, came from an environment in China where the m e a n s were in the same "stone age" in which I had started: she knew how to cast dialysis bags from collodion, how to carry out quantitative transfers, a n d how to do weighings on a microbalance. As a consequence, she was able to do experiments at the highest level of difficulty, solvent interaction m e a s u r e m e n t s at high t e m p e r a t u r e where evaporation becomes a major problem. All of this may reflect the disappearance from the Western undergraduate c u r r i c u l u m of gravimetric quantitative analysis a n d similar disciplines, which developed in the student the care a n d meticulousness n e e d e d in high-precision n o n i n s t r u m e n t a l quantitative work. I am concerned by the use of "kits" while ignoring their chemistry. I wonder how m a n y would be capable to distinguish an optical reflection from a real b a n d in a computer-controlled spectrophotometer or to correct properly for turbidity which can be m i s t a k e n for absorption: c o m p u t e r s do not know optics!

I Become an Academic: Brandeis

One day, while I was sitting in my Philadelphia office, the phone rang. To my "hello" came the reply: "Hi, this is Nate"; "Nate who?" "Nate Kaplan. Listen, do you want to come?" -"Come where?" -"Come to Brandeis; I am offering you a Professorship" This was a total surprise. A n d so in the fall of 1966, I left the sheltered environment of a government laboratory a n d Fate directed me into what I believed was the pure intellectual world of

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Academia, composed of m e n fully dedicated to the search for objective truth. As time went on I learned the labyrinthine politics that exist in the lofty academic world. I m u s t point out that I h a d met Nate only once, at a s e m i n a r that I h a d given at Brandeis a few m o n t h s prior to the call. At Brandeis, I struck friendships with several colleagues, in p a r t i c u l a r with Bob Abeles and Bill Jencks, two giants of biochemistry. Over the years we spent many hours talking. Occasionally, it was about science. With Bob Abeles our conversations centered on history, military history in particular, and politics. There were few m e n w h o m I a d m i r e d as m u c h as Bob for his absolute integrity a n d courage. Bobs n a m e belongs in "Profiles of Courage" He has e a r n e d this by his two struggles with life-threatening diseases. In the first one he u n d e r w e n t one year of chemotherapy without ever complaining and without relinquishing any of his duties as C h a i r m a n of the D e p a r t m e n t or missing a lecture. Fifteen years later he was faced with another debilitating disease. He came daily to the laboratory, obviously in great pain, almost to the end. Bob was absolutely objective in his judgments. He was totally anti-Soviet and anti-communist. In our conversations, he used to r e m a r k that, after all, Stalin had killed more people t h a n Hitler. He h a d enormous respect for great statesmen. I recall h i m telling me with anger of the occasion when he learned that a teacher in the local high school h a d qualified Bismarck of being an idiot. Bob s t o r m e d into the Principal's office a n d d e m a n d e d an explanation of how such an incompetent could be teaching. D u r i n g our conversations about the Second World War as a military operation, he expressed his admiration of the G e r m a n generals. His travels took h i m frequently to Germany. W h e n he had the possibility of visiting one of the concentration camps, he refused to go. As he said to me he saw no reason why. I learned only at his funeral that, as a child in Vienna, he h a d to wear a yellow star. With Bill Jencks the topics of conversations at our weekly lunches were music, F r e n c h romanesque architecture, a n d

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Byzantine culture which fascinated him. Without s p e a k i n g a word of French, Bill traveled to r e m o t e corners in Prance by bus and secondary rail connections to see some p a r t i c u l a r church or statues t h a t he had read about. Some of these itineraries were probably firsts in history. One day, d u r i n g one of our lunches, Bill remarked: "Do you w a n t to know how I found God?" Bill was a great admirer of Bach and he regarded the Bach partitas as absolute perfection in music. In reading about Bach, he found a quotation: when Bach was asked how he wrote such perfect music, he answered: "I write this for G o d " A n d Bill concluded: "Now I know who is God. God is someone for whom one writes perfect partitas" In science Bill Jencks h a d the gift of shocking me out of m e n t a l lethargy into an intense t h i n k i n g process by springing an on the surface totally naive question. One day, early in my t e n u r e at Brandeis, as we were r e t u r n i n g from one of our noontime walks, he asked: "What is binding?" I was flustered: what a strange question! The question stayed with me. In fact, this is what I have been t h i n k i n g about for four decades as I was developing linkages, t h e r m o d y n a m i c boxes, as well as the t h e r m o d y n a m i c s of preferential binding, exclusion, a n d hydration. My single difficulty with Bill was to convince h i m that enzyme active sites h a d to be looked at in t h r e e dimensions. All biochemical m e c h a n i s m s were always two-dimensional. Then, one day, Bill discovered s t r u c t u r e on his own and we never had to discuss the point again.

Jeffries W y m a n About that time, I also m e t Jeffries Wyman. Scientifically, I was his absolute disciple: the Wyman law of linkages [33,34] became central to my analysis of interactions at all levels; it opened the way to u n d e r s t a n d the controls in protein assembly, ligand binding, and solvent control of conformation. The Wyman theory of linked functions can be s u m m a r i z e d as follows: any biological

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system can be regarded as a three-dimensional mesh of chemical reactions. Each strand in the mesh is a linkage in the Wyman sense. The entire organism is an enormous such mesh. Clean isolation of a reacting system (for example, by chemical isolation of its components in a test tube) makes it possible to establish the linkages within it. Linkage means coupling between two reactions, such that one cannot proceed without the other. The simplest linked system is found in the modulation of a reaction by a ligand, e.g., activation of an enzyme by a cofactor, urea denaturation of a protein, ion-induced self-association of a protein. The linkage equation for a reaction, P ~ P* (modulated by a ligand, L), is:

(dlogK/dlogaL) = vp ' - VL P = AVE where aL is the thermodynamic activity of ligand L and PL is the thermodynamic binding of ligand L to the protein. The Wyman equation shows that, if a ligand affects an equilibrium, there must be a change in the extent of the thermodynamic binding of the ligand to the protein during the course of the reaction. The plot is:

Av L

\

log a L (conc L)

This plot shows that the slope at any concentration of ligand is the change in ligand binding at the given ligand concentration (it can be positive or negative). Tanford [35] later extended this to show that measured A v L is, in fact, the difference between changes in the numbers of effective water molecules (rw) and ligand molecules (rL) which interact thermodynamically with

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the protein, without necessarily forming direct contacts, A VL= ArL--(mL/mw) Arw (where mL and mw are molal concentrations of ligand and water). Simply, this means that in a mixture of water and ligand (the protein environment), space vacated by one must become occupied by the other (there can be no vacuum). We have applied systematically the Wyman theory to the characterization of a variety of systems: the linked equilibria between tubulin, double rings, and microtubules and the factors that control them, Mg 2+, Ca 2+, GTP, GDP; the interactions between tubulin and colchicine and the vinca alkaloid drugs; the stabilization and denaturation of proteins by weakly interacting solvent components, such as sucrose and urea. Jeffries befriended me.We took many walks together at various scientific conferences, be they on a Greek island, the Italian Riviera, or simply New Hampshire. Jeffries would talk about

Fig. 1.

Constructing a thermodynamic box at my orderly desk.

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Fig. 2. Second [~ternclti(,~cd Con#~rc~ce o~ [3i~H~ern~ody~amic.s. Seg[4cm, Austria, 1985. :l/htrine Gorbunoff ctnd (;io~'cmT~i l-~icddi ctre cdl clttention to some fine point made by Chclrles Tc~n/brd, zLhi/c S S ' T is e12gro.~sed i~7 ]Tis own meditations.

some of his amazing travels, but, with me, very much was about the Russian Revolution, a topic that fascinated him. In later years, when Jeffries became immobilized, my wife and I used to visit the Wymans in their country home in Burgundy, near Sens. Jeffries and I would sit under a plum tree, drink tea, and talk, but now he was reminiscing about his childhood in Wellesley, where we were living. Indeed, it h a d been a different world! All this was u n d e r the watchful care of his genteel aristocratic Russian wife, Olga Alexandrovna, who adopted us as her children.

Two Encounters at Santa Margherita Ligure One of the rewards of being a protein physical chemist was invitations to participate in conferences on protein thermodynamics which were invariably held in pleasant surroundings.

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Particularly memorable ones were those that Giovanni Rialdi organized in Santa Margherita Ligure. It was there that, in 1976, I met Hans-Jurgen Hinz, a young G e r m a n physical chemist, who was a specialist in protein microcalorimetry. Soon we were measuring together in Regensburg the heat effects associated with tubulin self-assembly into microtubules, a collaboration that evolved and continued when Hans moved to Muenster and continues to this day. And so, after many years away from the bench I came back to the laboratory working postdoc hours, because all tubulin experiments take a full 12-h day. During these long hours we would c a r r y on a conversation about the state of the world and, in particular, the West's docility before communist regimes. Hans had spent several months in Pushchino. He saw there the reality of life in what he called "the worker's paradise:' This was never to change. In this he reminded me of Bob Abeles. Having obtained one more heattransition curve on tubulin self-assembly, we would come to a

Fig. 3. A chance encounter in Regensburg (1990) between four protein thermodynamicists. Left to right: Stan Gill, Peter Privalov, Serge Timasheff, and Hans Hinz.

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leisurely dinner in the w a r m atmospheres of the Hinz home. His wife, Barbara, is an excellent cook and Hans always has exquisite wines. I do not know anybody who understands, as well as Hans, the equilibrium and nonequilibrium aspects of calorimetry and their relations to protein thermodynamics. Hans h a d great difficulty in getting accepted his criticisms of the ubiquitous simplistic interpretations of calorimetric data. At times, he was furious about this. One of my most i m p o r t a n t contributions in our collaboration was in helping h i m to find a formulation t h a t would be understandable to others a n d deliver his message [36]. Alas, he acquired few followers. With Hans and Barbara, my wife and I share many principles and interests. It is always delightful to spend a few days in their company, be it in Muenster or Paris. Being Russian a n d inbred with Russian culture from childhood, I always had an interest in w h a t is the reality of life in that country under the Soviet regime. I h a d h e a r d enough stories from members of the Second Emigration t h a t led to a feeling of compassion for those living u n d e r that oppressive system. The few chance encounters which I had with Soviet people, mostly at scientific meetings, left me with a definite impression that they preferred not to talk with me, or rather were afraid to do so. In any conversation, even casual, it was obvious that they were weighing every phrase, every word. At times, in a museum, h e a r i n g us talk in Russian, someone would accost us and ask in Russian: "Where are you from?" On h e a r i n g "Boston,' they would start and, with some mumbled r e m a r k about continuing their visit, would wander off. Obviously, "White Russians" were not desirable company. As a consequence, I never sought encounters with Soviet people. A n d then, at the 1976 m e e t i n g in Santa Margherita Ligure, I met a m a n who was totally different. It was Peter Privalov. The first evening at dinner, we h a p p e n e d to sit facing each other. I did not know who he was other t h a n a Soviet scientist. After a polite greeting, I c o n t i n u e d to talk with whomever sat next to me. W h e n we stood up from the table, Peter Privalov walked up

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to me, introduced himself, and suggested t h a t we take a walk outside. As I was t u r n i n g toward the marina, he indicated the other direction a n d we went off strolling t h r o u g h some backstreets where, as he remarked, nobody could hear us. Peter was very open about life in the USSR. He was very interested in Russian history, in p a r t i c u l a r the time of the Revolution, and he would ask me m a n y questions, especially about the role played by various individuals. He is one of the rare Soviet people who know Russian history. In earlier encounters with others, I h a d found t h a t most, but not all, events t h a t h a d t a k e n place two centuries back were p e r m i t t e d topics. There were gaps in their knowledge, because censorship reached that far back. (One has to r e m e m b e r that the second p a r t of Eisenstein's film "Ivan the Terrible" was not released: the scenes of his "terror" r e m i n d e d too m u c h of the t h e n c u r r e n t Stalinist reality.) No one h a d h e a r d of the freeing of serfs by Alexander II in 1860, a few years before Lincoln's E m a n c i p a t i o n Proclamation. No one k n e w of. Stolypin's great reforms in the early 20th century. Stolypin was k n o w n as the Tsar's h a n g m a n and oppressor of the people (a few terrorists were executed after trial following the 1905 Revolution). Peter knew all of this. He had read Dostoyevsky (Dostoyevsky was on the forbidden list). Peter knew about the February Revolution (which was a revelation to m a n y after the collapse of the regime in 1990). He had realized that the evil lay not in Stalin, but in Lenin. He also told me about life there: as a scientist he did belong to a privileged class but life was dull and the atmosphere stifling. W h e n I comm e n t e d on the i m m e n s e a m o u n t of data he h a d accumulated, he simply remarked: "In Pushchino, one can work, d r i n k or h a n g himself. I chose to work:' Later, t h r o u g h the efforts of A m e r i c a n a n d E u r o p e a n colleagues, he came to the U n i t e d States to give some lectures. I a r r a n g e d for a s e m i n a r at Brandeis. To protect him, the invitation came from one of my colleagues. He stayed with us over the weekend. He spent the whole time r e a d i n g memoirs of Russian revolutionaries who had k n o w n Lenin, etc., books totally forbidden in the Soviet Union. Peter was

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perfectly aware of the n a t u r e of t h a t regime, its oppression, a n d corruption. Whenever we met at conferences, he gravitated t o w a r d us a n d we would go for walks, s p e a k i n g in Russian, a n s w e r i n g his questions, a n d listening to his tales. Finally, the regime collapsed and, since then, we have been able to t a l k freely in the c o m p a n y of others.

B e h i n d the Iron Curtain My sole excursion behind the Iron C u r t a i n was in 1978, w h e n I was invited to the E u r o p e a n Congress of Biophysics in Dresden, East Germany. At the West Berlin airport, we were loaded onto East G e r m a n buses. As we were approaching the Wall, I could see a large pair of binoculars focused on the bus from a high tower. The bus m e a n d e r e d t h r o u g h the barricades inside the Wall a n d came to a stop in front of a barrier. There was silence. Ten m i n u t e s later, the door of a b a r r a c k opened a n d a young m a n in u n i f o r m came out. He opened the front door of the bus, entered and s t a r t e d t a k i n g passports from passengers. He would look at the person, t h e n at the i.d. photo, repeat this several times, t h e n s t a m p the passport a n d h a n d it back. Not a word was spoken. His face was expressionless, wooden. I was in the second row. He took my passport, did the exercise, a n d p u t the passport in his pocket. A totally physical fright came over me: I was in East Germany, I h a d a Russian name, and no document. A h a l f h o u r passed, n o t h i n g was happening. It was a hot s u m m e r day. Someone opened the door of the bus to get some air; a m a n came a n d shut it from the outside. Finally, the door opened a n d a n o t h e r one came aboard. He h a d in his h a n d a n u m b e r of passports. He would open each, look at the i.d. photo, t h e n walk up a n d down the bus looking at people's faces a n d suddenly t h r u s t t h a t passport at someone. Finally, I got mine back. Some fool at the East G e r m a n consulate in Washington h a d neglected to s t a m p it somewhere.

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On arrival in Dresden, I was s t r u c k by seeing street n a m e s in Russian, with G e r m a n in smaller letters. The m a i n square was d o m i n a t e d by a huge statue of Lenin. I b o u g h t two postcards of it a n d sent t h e m to my wife a n d to Bob Abeles addressed: "Dear Comrade!" My talk was scheduled for the second day of the meeting, July 4. I was the first on the program. W h e n I was introduced, I opened w i t h the words: "The topic t h a t I should like to talk about today, on the 202nd anniversary of the A m e r i c a n Revolution, i s . . . " The r o o m broke out in an ovation. I h a d provoked a political incident. The word Revolution was sacred a n d referred to only one: Lenin's coup d~tat in October 1917. By t h a t time everybody h a d become totally fed up with the r e g i m e n t a t i o n a n d inefficiency to which we were subjected. Later, a n u m b e r of East E u r o p e a n scientists approached me w i t h approving remarks. The next day I was scheduled to chair an a f t e r n o o n symposium. Peter Privalov was the last speaker. That m o r n i n g I found an envelope with an invitation to a reception in Meissen. The bus was leaving at 4:30 pm; the session was e n d i n g at 6:00. In a q u a n d a r y I went to the General Secretariat. The very friendly East G e r m a n s (who all spoke Russian) were totally perplexed about w h a t to do. One of t h e m finally said: "Do whatever you would do in your country:.' So, I asked one of my friends to chair the last p a r t of the program. I arrived well in advance. Suddenly a m a n walked up to me a n d said: "I am XYZ (a well-known m e m b e r of the Akademiya Nauk). All is arranged. Privalov's t a l k will be first; after the second t a l k ABC will take over the chairmanship. You will b o t h get on the bus" It was evident who was in charge. On the way to Meissen, Peter a n d I t a l k e d about Russian opera a n d c u r r e n t singers. In Meissen, after a visit of the porcelain museum, we went to the reception. There was a m o u n t a i n of food ( i m p o r t e d from West Germany). I noticed that I was one of the very few w e s t e r n scientists present. The crowd was all Soviet a n d East G e r m a n scientists. We h e a r d a speech given by a Politburo member. The t h e m e was how the "socialist" environment was p r o m o t i n g a flourishing of science, while it was l a n g u i s h i n g in the capitalist

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countries. One of the Soviet scientists r e m a r k e d to me: "It is so boring, we have h e a r d all of this m a n y times before." We sat at table. Then, the reason for my invitation became clear. I was s u r r o u n d e d by Russian scientists. All, very friendly, were u r g i n g me to come to the USSR and give lectures. As I h a d done on prior occasions, I refused politely. This was on principle: there was no compromise w i t h the Soviet regime. (A Russian acquaintance of m i n e did accept in a w e a k moment. He was i n t r o d u c e d at t h e lecture with: "His father was a traitor to his country. The son has r e p e n t e d a n d now he is here:') I was i n t r o d u c e d to the vice president of the Academy of Sciences. He was a cultured, affable m a n (he h a d spent t e n years in the Gulag). He invited me on behalf of the Academy. I declined again, saying: "I shall not go as long as your government violates the Helsinki accords" T h e n one m a n (it was XYZ) exclaimed: "Then you will never come!" He was wrong. Twelve years later, the r e g i m e collapsed a n d I have been, b u t not to give lectures. I left Dresden by t r a i n for Berlin. This provided the final taste of "workers' paradise": the official at t h e rail ticket booth in Dresden was refusing to accept my East G e r m a n m a r k s (the currency in which my expenses h a d been reimbursed); he was d e m a n d i n g US dollars because I h a d an A m e r i c a n passport. Finally, after a 1-h long a r g u m e n t , I prevailed a n d he did take the c u r r e n c y of his country.

My Talented Postdoctorals At Brandeis, I was blessed w i t h a succession of t a l e n t e d postdoctorals. My laboratory became a little UN, as postdocs came from Japan, India, China, Taiwan, Spain, Chile, Australia, Germany, a n d France. Those from N o r t h America were a small minority. Over the years I learned t h a t pedigree (the prestige of the i n s t i t u t i o n whence they came) h a d little influence on success. W h a t was required was intellectual commitment,

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perseverance, and humility. I shall talk about some of them, in as much as this will depict the general philosophy of my laboratory, the evolution of the central ideas, and of the approaches that were adopted for the study of various interacting systems. All along there were two main themes - protein-self-association and protein-solvent interactions. I do not intend to describe systematically our research; it is documented in the literature. I shall describe, however, the logical development of the problems and the thread laid out for us by the systems themselves. Two of the earliest postdoctorals were Kirk Aune and Dick Weisenberg, both of whom had a lasting impact, each in his own way. Kirk Aune had just completed in Tanford's laboratory an in-depth study of the guanidine hydrochloride denaturation of lysozyme,* which saw the first application of the Tanford decomposition of the Wyman linkage equation into changes in the interactions of water and ligand molecules with the protein during the course of the reaction [37]. When he arrived in my laboratory, Kirk was a fully developed scientist. He had a deep understanding of protein thermodynamics and he was not easy to convince of any suggestions. We had many a lively discussion. Kirk came to my laboratory because he wanted to study a selfassociating system and was well familiar with our work on fl-lactoglobulin. I suggested to him the acid pH dimerization of a-chymotrypsin, which was of general interest at the time. In well-thought-out sedimentation equilibrium experiments followed by analysis of charged group interactions by electrostatic theory, Kirk established definitively that the specific interaction that held together the two molecules in solution was a pair of symmetrical ion pairs between His 57 and the a-COOH of Tyr 146, in fact the interaction seen in the crystal unit cell [38]. He then showed in experiments as a function of NaC1 and CaC12

*See also in this series C. Tanford's autobiographic chapter (Vol. 42, i.e., VII of the "Personal Recollections" G. Semenza and A.J. Turner, Eds., 2003; pp. 1-52) and the chapter by J.T. E dsall on his scientific life and on that of J. Wyman (Vol. 36, i.e., II of the "Personal Recollections;' G. Semenza, Ed., 1986; pp. 99-1959). (Note of the Editor.)

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concentrations and application of the Tanford expansion of the Wyman equation that the dimerization process was accompanied by the departure of a minimum of 250 effective water molecules from the surfaces of contact [39]. This was the first application of the Wyman-Tanford theory to an interaction of a native globular protein. Many others were to follow in my and other laboratories. The interest of this story lies mainly in its corollary. Like many families, the chymotrypsin family has a skeleton hidden in the closet: there are two isomers, a and • with identical amino acid sequence (identical twins). Both are generated from chymotrypsinogen by excision of the same two dipeptides, one of the excisions leading to Tyr-146 becoming a C-terminal residue, the one involved in the dimerization. These two isomers are prepared by different pathways of activation of chymotrypsinogen in which the two dipeptides are excised in different order. The then held dogma was that tertiary structure is absolutely determined by primary structure. The primary structures of the two being identical, the necessary conclusion was that a- and y-chymotrypsins in solution must be indistinguishable. The X-ray crystallographic structures of the two had been determined (in those days each appearance of a three-dimensional structure was an event): there were small differences in the orientations of Tyr-146 in a- and y-chymotrypsins [40]. However, the two had been crystallized out of different media, and a small surface difference within the crystals could easily be ascribed to different modes of crystallization. At the time, M a r i n a Gorbunoff, who was studying the surface exposure of tyrosine groups by chemical probes, found that one of the tyrosines was exposed to different extents in a- and y-chymotrypsins [41]. I suggested to Kirk that he should look at the dimerization of y-chymotrypsin. His reaction was: why waste the time? After all, Tyr-146 was a terminal residue located on the protein surface and the dogma required that, in dilute buffer solution, it would find rapidly the position of minimal free energy. After several days of lively discussion, Kirk agreed to do the experiment.

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Two days later, he came to my office and asked calmly: "How did you know?" (the dimerization of y-chymotrypsin was much weaker t h a n that of a). I answered: "Kirk, I have been talking with proteins for twenty years:' He shook his head; I had just told him about intuition, which briefly can be described as knowledge acquired beyond the sum total of the observations. In the case of chymotrypsin, as Kirk concluded, structural constraints present in the parent molecule had obviously been released to different extents in the two sequences of dipeptide excision, making it impossible for even a surface terminal residue to move freely. In sociological studies, it is known that identical twins raised in different environments will not necessarily display identical characters. This leads to the First Law of Molecular Sociology: The behavior of molecular identical twins is conditioned by their early upbringing. How did I know? Very early in my career, I had learned to "listen to the protein" and let it guide me, i.e., to use intuition. This meant never to be dogmatic about following a preconceived course, but always to look beyond the data, and be ready to follow the leader into an unexpected direction. There is a great fallacy in what is commonly called the "scientific method:' which consists of stages of data gathering, data analysis, and conclusion. New knowledge is never acquired in this way. (I have seen scientists at ERRL following this approach seriously; they never made any significant discoveries.) The establishment of new "truths" requires thinking beyond the observations all along the research process. This topic is developed in-depth in the epistemological theory of intuitivism of the great philosophers, Nikolai Lossky and Henri Bergson. In fact, I had frequently heard Kirkwood's r e m a r k about the needs of intuition and humility in the advancement of science. One must never be arrogant and make assertions based on convictions (dogma). One must have the humility to question these constantly, to admit faults in one's reasoning, and to correct them in accordance with what "the system is telling," i.e., by constant application of an intuitivistic process. Kirkwood also used

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to r e m a r k on the aesthetics a n d elegance of the solution of a problem. And, indeed, in my experience, what seemed to be complex c u m b e r s o m e processes, would unravel to simple aesthetically appealing pictures. Kirk was i n s t r u m e n t a l in my getting two excellent researchers. Shortly before he left he r e c r u i t e d a superb graduate student, R o n Frigon (one of the two t h a t I h a d in all my years at Brandeis) and, later, w h e n he was a Professor at Baylor University, he passed on to me a postdoctoral from India, V. Prakash. Kirk told me t h a t P r a k a s h was a n amazingly organized a n d careful laboratory researcher who p r o d u c e d absolutely reliable results at a consistently rapid pace. He was right. I shall t a l k about these in the proper places. Shortly before Kirk left, among applications for postdoctorals, one came from J i m Lee. I invited h i m for an interview. W h e n he came, I found a very modest young man. We t a l k e d about his experience. He had some with light scattering, but it was evident that he was not at all p r e p a r e d to do the k i n d of work done in my laboratory - Wyman linkages, m u l t i c o m p o n e n t light scattering, p r o t e i n self-association with in-depth thermodynamic a n d hydrodynamic analyses. In fact, few were p r e p a r e d to do this. There were some exceptions, for example my two Australians, L a r r y Ward a n d Keith Shearwin, b o t h of w h o m were students of Don Winzor, a superb p r o t e i n physical chemist in Brisbane. As usual, I t u r n e d J i m Lee over to my postdocs w i t h w h o m he spent a few hours. W h e n he r e t u r n e d to my office at the end of the afternoon, he said with great humility: "Dr. Timasheff, please let me come to your laboratory and learn:' This p e r s u a d e d me to accept him. W h e n J i m Lee arrived he chose to study the interaction of t u b u l i n w i t h vinblastine. As was my practice, I did not p u s h h i m to produce results, but I essentially left h i m on his own to learn. We t a l k e d whenever he wished. I would walk in daily into the laboratory a n d ask a general question: "How are t h i n g s going?" The postdocs k n e w t h a t it was an invitation to discuss the work. This is also w h e n they told me of laboratory needs.

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My practice was to start them off and, then, let them go at their own pace. I was always available to answer questions. I intervened only when it was obvious that someone was going off into "the woods" or getting bogged down in trivia. My involvement would become major in the late in-depth analysis and finally in the writing of papers. Writing of papers was a slow process. Papers would evolve through several versions, with resting periods in between. It is amazing how well one sees inadequancies on picking up a manuscript after a few weeks. I was never concerned about rapid publication. I was concerned about not introducing errors into the literature. I knew that in the solvent interaction field we were alone and in the microtubule field nobody would apply the rigorous physicochemical analysis that we did, although the methods were inherently not difficult (three-component thermodynamics, Wyman linkages, Gilbert analysis of sedimentation patterns, etc.). Others were rushing to publish before someone else did. (As a consequence, microtubule-tubulin literature soon earned the reputation of being 90% a jungle of unverified claims.) Jim Lee stayed with me for six years (most good postdocs stayed more t h a n two years). He spent the first year learning techniques, both laboratory and analytical, theoretical, and also learning how to think quantitatively as a physical chemist. At that time, K r a t k y and Porod in Gratz had developed their high-precision solution density meter and it became available commercially. This was obviously ideal for measuring preferential binding to proteins of weakly interacting solvents. Such studies require the ability to measure in a dialysis equilibrium experiment solvent component concentration differences of the order of 0.01 mg/mL between two solutions with concentrations of ~100 mg/mL, i.e., one part in ten thousand. Use of the densimeter permitted to do this. So, we bought an instrument. Jim Lee got interested. He interrupted his work on tubulin-vinblastine interactions and set out to develop the necessary techniques. He established conditions of absolute stability of measurements, developed techniques for the quantitative evaporation-free transfer of solutions

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from the dialysis bag into the cell, concentration measurements, etc. He then proceeded to measure the preferential interaction of 12 proteins denatured in 6 M GuHC1 and found that, at that concentration, all exhibited a very low preferential interaction with the denaturant [42], confirming the conclusion reached earlier by Hade and Tanford [43]. The four proteins that overlapped in the two studies gave identical values. Hade and Tanford had done their measurements by isopiestic vapor-phase equilibrium. This coincidence of values obtained by totally unrelated techniques reassured us that the developed procedures gave reliable results. They were followed, with some occasional modifications, in the extensive solvent interaction studies in my laboratory for the next 25 years. Jim Lee was now ready and, as we shall see, he solved two major problems before going on his own. Since then, he has done remarkable physico-chemical studies of controls in subunit enzymes and other biological systems. In these he has developed linkage analyses that go far beyond any that we ever did.

Tubulin, Double Rings, Microtubules Dick Weisenberg came from Ed Taylor's laboratory, where he had purified and characterized tubulin, the protein subunit of microtubules [44]. Tubulin is a stable dimer of two similar, but not identical subunits, ~ and fl; it has a molecular weight of 110,000 and is characterized by a sedimentation coefficient of 5.8 S. Dick wanted to work on a protein self-association problem. H e told me about tubulin and his early observations in Taylor's laboratory that magnesium ions and the anti-mitotic vinca alkaloids induced aggregation in tubulin. I suggested that this would be an excellent system and Dick set out to look at its self-associations. He started by perfecting the purification process, because the then procedure left 5-10% impurity. This became known as Weisenberg tubulin, which

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we used with m i n o r modifications for the next 25 years. Most other laboratories used a different p r e p a r a t i o n procedure, which gave at best 95% pure tubulin. Using the purified protein in ultracentrifugal studies, he defined the general modes of t u b u l i n self-association in the presence of Mg 2+ ions, vinblastine, a n d colchicine [45]. All this required detailed studies to arrive at the exact characteristics of each self-association. W h e n he left, to my inquiry, he told me t h a t he was not interested in p u r s u i n g f u r t h e r any of these processes a n d t h a t we were free to do w h a t we wanted. A n d so, shortly after, my graduate student, Ron Frigon, s t a r t e d the detailed characterization of the m a g n e s i u m - i n d u c e d polymerization, while J i m Lee took on the vinblastine system. Weisenberg had an amazing intuitive u n d e r s t a n d i n g of tubulin. Over the years, he was coming up w i t h insightful ideas about the system, most of which t u r n e d out to be true. His problem was to devise clear-cut experiments to test them. His major achievement was the r e c o n s t i t u t i o n of microtubules from partially purified t u b u l i n [46]. He showed t h a t this process was inhibited by calcium ions, but p r o m o t e d by glycerol a n d GTP, a n d t h a t the GTP was hydrolyzed to GDP subsequent to polymerization. At the time, there was a race to do this and, deservingly, Dick won. Dick Weisenberg also identified the 24-28 t u b u l i n double rings, which will be discussed presently, as the storage form of the protein [47]. Years later we were able to confirm this concept. The magnesium-controlled self-association of t u b u l i n was the topic of R o n Frigon's PhD thesis. Ron was very i n d e p e n d e n t a n d he did not want me to interfere with his research. His s t a n d a r d r e m a r k was: "Doc, w h e n I am ready to talk, ~ shall k n o c k on the door:' A n d on occasion, he did. The work was done by velocity s e d i m e n t a t i o n as a function of Mg 2+ ion concentration a n d in the presence of GTP. The Gilbert-type patt e r n s obtained indicated a process of linear g r o w t h of a t u b u l i n chain t h a t was t e r m i n a t e d by a ring-closing step. In a sophisticated analysis of the hydrodynamic data, Ron showed

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that the best description of the final polymer formed was a closed-ring structure that contained 26 a - ~ tubulin monomers. To verify his conclusion, Ron looked at these aggregates in the electron microscope. The structure found was a double ring, made up of 26 • 4 a - ~ tubulins with outer and inner diameters of 47 + 3 nm and 27 =t:3 nm, respectively. To test whether the rings correspond to the polymer observed in sedimentation velocity, Ron calculated their expected sedimentation coefficient by using the general hydrodynamic theory of Kirkwood. He found a value of 43 • 3 S which agreed with the experimental value of 42 S [48]. A Wyman analysis of the involvement of Mg 2+ ions showed that the addition of each a-fl tubulin monomer to the structure was accompanied by the binding of one Mg 2+ ion, which t u r n e d out to be an important switch in the tubulin-microtuble system. In this study the question was also addressed: why is the system pulled to the final structure, namely the closed ring with little evidence of any intermediates? This was addressed by the analysis of the thermodynamics of the addition of the last, in this case the 26th subunit [49]. The first 25 subunits add sequentially in chainlike fashion with identical association equilibrium c o n s t a n t s , ka. Addition of the last subunit (shown schematically in Figure 4) involves the closing of two intersubunit bond. The first bond is simply an addition to the chain, with equilibrium constant ka. The second bond, however, which is ring closure, is a unimolecular reaction while all the growth steps are bimolecular. It consists of the formation of an intertubulin contact, characterized by ka, with no decrease of the total number of particles in solution, and hence no loss of entropy of mixing, known as the cratic entropy. This contributes -2.5 kcal/mol to the standard free energy. Then, the association equilibrium constant for the formation of the last bond is kay , where y represents the contribution of various entropic terms, and the full binding constant for the last, 26th tubulin monomer is kn--k2ay. Generally, k n ~ k 3. As a consequence, once polymerization starts it is pulled overwhelmingly to the

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ka

A

+ A

A

~-A

jA

Fig. 4. Why the final closed "ring" is the overwhelmingly predominant structure. The addition of the final subunit is broken down into two hypothetical steps. First, the subunit, A, adds to the growth end of the chain with a growth equilibrium constant, ka. In the second step, the final intersubunit bond is formed closing the ring; this step consists of bond formation, k~, which, in this step, is a unimolecular reaction, whereas it is a bimolecular reaction in all the preceding, chain-growth steps. Since there is no change in the number of particles, there is no loss of the entropy of mixing which accompanies all the preceding growth steps; this contribution of the free-energy gain is represented by y. Therefore, the addition of the final subunit occurs with an equilibrium constant k2ay, which pulls the entire associating system overwhelmingly to the final closed structure.

final stage w i t h intermediates being present only in m i n u t e amounts. Having fully characterized the Mg 2+ ion-controlled r i n g formation, we t h o u g h t t h a t our involvement in t u b u l i n self-association h a d come to an end. Fate h a d decreed it differently. Late in Ron's study a n u m b e r of papers s t a r t e d appearing on the formation of microtubules in vitro (reviewed in Ref. 50). All of the studies were done with partially purified tubulin, at best 95% pure. There was obviously a r u s h to publish a n d little critical thinking. All the "tubulins" used displayed a n u m b e r of extraneous protein b a n d s on gel electrophoresis. Claims were made that certain of these proteins, M A P s (microtubule associated proteins), or v (the protein essential for microtubule assembly), were required for microtubule assembly; preexisting nucleation centers were "identified"; the formation of microtubules in the presence of buffer ions with strong dipole m o m e n t s led to proposals of physiological

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regulation by zwitterions, etc. In one respect there was universal agreement: all maintained that a-fl tubulin monomer (5.8 S) was absolutely unable to polymerize into microtubles in the absence of whatever of the claimed microtubule assembly controlling agents. All of these proposals were based on the solid scientific approach of wishful thinking. There were signs of neither intuition nor humility, and the patterns were far from aesthetic. Among all this mountain of publications, one report attracted our curiosity. In it, the products of the cold depolymerization of microtubles were examined by electron microscopy. The products were spiral-shaped structures and double rings very similar to those just found by Frigon as the 42 S terminal product of the reversible self-association of pure tubulin in the presence of magnesium [51]. It was stated, however, that these rings are not in equilibrium with the 5.8 S u-fl monomer, which led to the proposal that the 5.8S tubulin must be chemically different from the tubulin found in the rings formed on the cold depolymerization of microtubules. This statement puzzled us: Ron had just made rings from that very 5.8 S tubulin. At this point, my Jesuit training in logic took over. The syllogism was: (1) If microtubules can disassemble reversibly to rings; (2) and if rings, as shown by Ron Frigon, are in equilibrium with pure u-fl tubulin; (3) then microtubules should form from pure tubulin. What was needed was an experimental test. I went to Ron, but he had just defended his thesis and was tired. Jim Lee was busy at that time with his Gua. HC1 work, but he agreed to do the experiment. What was needed was the correct protocol. Microtubules were known to be stable at 37~ but to depolymerize in the cold. So, we dissolved "Weisenberg tubulin" in the medium in which Ron had made reversibly double rings and modified it according to what was k n o w n to stabilize microtubules, i.e., we added 3A M glycerol and 10-3M EGTA (a calcium ion-sequestering agent). Jim Lee introduced this tubulin solution into a spectrophotometer, set at 350 nm (an optimal wavelength for following turbidity), and heated the sample to 37~

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Following a 2-min lag time the turbidity went up until it reached a stable plateau. Cooling to 20~ reversed the process: the turbidity r e t u r n e d to zero. Several such cycles were performed. We were making large structures in reversible manner. Addition of calcium ions prevented the process. The assembled material was then properly cast on a grid and examined by electron microscopy. The pictures showed filaments identical with published electron micrographs of microtubules. In less t h a n one hour we had made reversible microtubules from pure 5.8 S tubulin monomer [52]. All the previous dogma had to fall. No preexisting nucleation centers nor promoting proteins were needed, all the tubulin molecules were able to enter reversibly into both microtubules and double rings. (MAPs and v were found later to be important physiological molecules, but they were not involved in controls of microtubule formation.) Our electron micrographs of microtubules always also contained double rings showing that the two assembly processes could proceed in parallel. The significance of this became evident only 15 years later. Jim Lee followed with a detailed study in which he showed that assembly proceeded equally well in a variety of buffers, as well as in the absence of glycerol. In fact, in vitro, tubulin self-assembly into microtubules t u r n e d out to be thermodynamically an amazingly simple process. The only requirement was to have pure protein [53]. This reminded me of a remark made by a seminar speaker when I was a graduate student: he had just shown the major effects of minute amounts of impurity on the critical concentration of micelle formation by detergents and concluded: "cleanliness is next to godliness" an old English saying. Following this demonstration, Jim Lee proceeded to a thermodynamic analysis of the system. The process of microtubule assembly can be treated best as a nucleated polymerization [54], characterized by a critical concentration, C c r i t , a t which the large assembly starts growing from small, difficult to form, nuclei. The equilibrium constant for the addition of each

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unit (a-fl tubulin) to the growing structure is Kg =Ccrit. -1 While this thermodynamic presentation is simple, kinetically the process has been shown to be complicated (reviewed in Ref. 50): the rates of assembly and disassembly at the two ends of a microtubule are not identical, there is treadmilling of the subunits along the structure, the GTP on adding u-fl tubulin subunit becomes hydrolyzed to GDP after assembly, and Ca 2+ ions speed up the hydrolysis process. Thermodynamics, however, are independent of kinetic pathways. Therefore, if conditions are established for maintaining a steady state (e.g., having a large excess of GTP in the medium, so that all dissociating a-fl tubulins in the GDP state immediately become converted to GTP tubulin), the observed pseudo equilibria can be treated formally as equilibria, which permits the performance of a consistent thermodynamic analysis. Using this approach, we characterized the assembly process thermodynamically. Van't Hoff analysis showed that the polymerization of tubulin into microtubules is characterized by positive changes in enthalpy and entropy and an apparent change in the heat capacity, A C p = - 1 5 0 0 c a l m o l - l d e g -1 [53]. The last value was confirmed exactly in calorimetric measurement in collaboration with Hans Hinz [55]. From these values we could conclude that the formation of each tubulin-tubulin contact was accompanied by the loss of "ordered" water, as would be true in the formation of hydrophobic contacts. The control of the reaction by Mg 2+ ions (and other ligands) was addressed by application of the thermodynamic box, which is the expression of linkages relative to a given reference state, say the absence of ligand. The basis of the analysis is the fact that the modulation of an equilibrium by a ligand, P + L ~ P'L, can proceed in two ways: (1)

P+L~-PL(K1);

PL~P*L(K2)

(2)

P ~ P* (K4),

P* + L ~ P*L (K3)

464

S.N. TIMASHEFF

This permits to construct a box, because KIK2 = K3K4. It is more convenient to express this in terms of the Gibbs standard free energy, AG ~ = -2.303 R T log K:

P+L

P*+L

""

N,

PL

i-

PL*

The reaction described by AG~ (and K4) is the protein transio tion in the absence of ligand. Since AG~ + A G2= AG~ + AG3,o measurements as a function of ligand concentration permit to deduct the contribution of ligand binding to the protein reaction relative to its strength in the absence of ligand. Analysis in terms of both the Wyman equation and the thermodynamic box showed that each step in the polymerization involves the binding of one magnesium ion. Following this, thermodynamic boxes of increasing complexity became a routine part of our analyses. A Wyman plot as a function of glycerol activity gave a slope of +1, suggesting the binding of one additional glycerol molecule to protein per tubulin incorporated into microtubules [53]. This binding, however, cannot be regarded as any kind of complex formation, because the glycerol is present at 1-4M, which would correspond to a binding constant of ~10 ~mo1-1. It only means enrichment of the protein environment in glycerol. Alternatively, this may m e a n the departure of ~14 water molecules from the immediate domain of the protein during subunit addition, which is fully consistent with the measured thermodynamic parameters. In fact, 14 water molecules are much too small a number for the surface areas of the contacts formed; this must mean that additional combinations of glycerol

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a n d water molecules m u s t leave the d o m a i n of the p r o t e i n until the full surface of contact is satisfied. The reason for the glycerol stabilization of microtubules will become clear w h e n I describe the solvent-interaction studies. After this initial solution of two f u n d a m e n t a l problems in t u b u l i n self-assembly, work w i t h microtubules stopped for essentially a decade. I never h a d a large laboratory. I preferred to work with four postdoctorals, a n d so choices h a d to be made. As a consequence, the p r o g r a m concentrated on protein-solvent interactions a n d on the m e c h a n i s m s of binding to t u b u l i n of colchicine a n d the vinca alkaloids, a n d their controls of t u b u l i n function. Eventually, all of the studies merged into a vast interm e s h e d domain.

Protein-Solvent Interactions: The Exclusion Principle The story goes back. W h e n Ron Frigon a n d J i m Lee s t a r t e d to work w i t h t u b u l i n they were faced w i t h a difficult situation. The Weisenberg t u b u l i n was stored in liquid nitrogen. Each day a vial would be t a k e n a n d thawed. There was always a precipitate which h a d to be centrifuged off. Yet, d u r i n g the course of the day, precipitate kept on forming slowly. This was an unacceptable situation. So, R o n a n d J i m came to see me w i t h the u l t i m a t u m : "We c a n n o t work w i t h this material, a way m u s t be found to stabilize it" I said: "Do w h a t biochemists do: - "Do you m e a n add 3 M glycerol?" I said: "Yes." So they went into the laboratory a n d before freezing the freshly p r e p a r e d t u b u l i n they added 1 M sucrose. From t h a t day on, t u b u l i n could be kept in a frozen state for months; all samples were clear on t h a w i n g and, after removal of sucrose by a 2-h c o l u m n procedure, the solution stayed clear in a n ice b a t h for the next 10 h [56]. This was enough for a postdoc's working day. At this point, J i m Lee came to me a n d asked: "Dr. Timasheff, now t h a t we have stabilized t u b u l i n with sucrose, please tell me how does it work?" I answered: "I don't know. Find out" A n d so, he did.

466

S.N. TIMASHEFF

At that time, had you asked any biochemist or biologist how do sucrose and glycerol stabilize isolated organelles or enzyme activity, the answer would have been that they probably form some sort of protective shell. In fact, no one had ever investigated this question, nor was anyone interested: it worked. Jim Lee, however, had the intellectual curiosity; and this led him to a major discovery, that of the exclusion principle. He took the most obvious approach which was to study the binding of sucrose to the protein by dialysis equilibrium with the use of the precision density meter. Tubulin was dissolved in a series of sucrose solutions, between 0.1M and 1M and dialyzed properly. The results were totally unexpected: the binding stoichiometry was negative, i.e., there was a lower sucrose concentration inside the bag t h a n outside: sucrose was preferentially excluded from the general domain of the protein, there was preferential hydration. Jim then obtained the same result with several other proteins [57]. The effect was universal. What does this mean and how is this related to protein stabilization? Thermodynamically, the binding observed at dialysis equilibrium, VL, is a measure of the perturbation of the chemical potential of the ligand by the protein, and vice versa PL ~

(0mL/0rnp)t~L:--(O~L/0mp)mL/(O#L/OmL)mp,

where m is the molal concentration, tt is the chemical potential (an expression of free energy), and L and P refer to ligand and protein. If the interaction is thermodynamically favorable, (O/tL/Omp)mLis negative and 1)L is positive; on the other hand, if it is thermodynamically unfavorable, (Ol~L/Omp)mLispositive, VL is negative, and the concentration of the ligand next to the protein is decreased. The experiments showed that for several proteins, including tubulin, interaction with sucrose is thermodynamically unfavorable; therefore, sucrose is preferentially excluded. How does this lead to stabilization of a protein, such

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as tubulin? Experimentally stabilization should manifest itself by a decrease of the d e n a t u r a t i o n equilibrium constant, K, for the reaction N ~- D w h e n sucrose is added. T h e n in the Wyman equation the slope, dlog K]dlog aL, would be negative, m e a n i n g t h a t va(Product) < vL(Reactant), i.e., binding to the d e n a t u r e d protein should be more negative t h a n to the native form. This was tested in reversible heat d e n a t u r a t i o n studies on two proteins (tubulin is not reversible) a n d it was found t h a t vL(Denat)--vL(Native) indeed was negative. Sucrose was more excluded from d e n a t u r e d p r o t e i n t h a n from native and so it p u s h e d the equilibrium t o w a r d native, t h u s stabilizing the protein. This led to the formulation of the Exclusion Principle:

Stabilization by cosolvents, i.e., solvent components which must be present at high (1-4 M) concentration, is by preferential exclusion: the cosolvent is more excluded from the unfolded (denatured) form of the protein than from the compact (native) form. In simple words this m e a n s t h a t the stabilizing action of cosolvents is exercised by their not coming into contact w i t h the molecule t h a t they stabilize. (This leads to the Second Law of Molecular Sociology:

Apartness is frequently a more favorable state than togetherness.) The Exclusion Principle was found to be a universal rule: it holds t r u e for all n a t u r a l osmolytes, amino acids, salting-out salts, a n d crystallizing agents, such as PEG. This finding brought a rare compliment, similar to one made m a n y years before by George S c a t c h a r d who referred to one of my papers as good work. At a conference on p r o t e i n stability organized by Buzz Baldwin, I t a l k e d about protein stabilization by sugars. Walter Kauzmann, who was the closing speaker, listed t e n questions t h a t he h a d w a n t e d resolved. One of t h e m was the cause of protein stabilization by sucrose. He t h e n crossed it out a n d said: "Serge has now solved this puzzle for me" W h e n I gave talks about this work, I found t h a t the Exclusion Principle was difficult to accept by biochemical audiences. Action by not coming into contact was not falling into the mainline logic: after all, biochemical reactions always involved the formation of complexes, e.g., enzyme-substrate, i.e., direct contact was required.

468

S.N. TIMASHEFF

Historically, preferential hydration, i.e., exclusion of the cosolvent had been invoked first by Pete von Hippel when he was examining the binding of neutral salts to polyacrylamide columns [58]. Now that we had the answer to the stabilization of tubulin by sucrose, we could ask the question: how does glycerol (1-4 M) enhance the formation of microtubules from tubulin? The answer came with the study of the interactions of proteins with glycerol, carried out by a postdoctoral from Japan, Kuni Gekko, a fully formed protein physical chemist. He showed in rapid order that glycerol is preferentially excluded from a number of proteins and, in reversible unfolding studies, he established the increments of negative binding on denaturation [59] (glycerol was being used in several laboratories as a stabilizer of tubulin). We knew from Jim Lees work that the addition of each a-fl tubulin monomer to a growing microtubule was accompanied by the effective increase of one glycerol molecule in the vicinity of the protein, but we did not have information on the preferential binding of glycerol to tubulin. This was provided by George Na, who found that VL was negative, i.e., glycerol was preferentially excluded from the tubulin [60].This meant that Jim Lees observation was an expression of the lessening of exclusion during self-assembly into microtubules - namely, that the thermodynamically unfavorable interaction between tubulin and glycerol was relieved by decreasing the surface of contact between protein and solvent by burying some protein surface in the intersubunit contacts in the aggregate. Therefore, the Exclusion Principle provided a universal explanation of the stabilizing action of cosolvents not only of the native structure of a protein (e.g., stabilization of enzyme activity), but also the integrity of assembled structure (e.g., stabilization of isolated organelles). At this point, the laboratory had an arsenal of solidly established approaches, both experimental and conceptual, and we were ready for further in-depth examination of tubulin self-associations and solvent interactions. The need for new talent was fulfilled with the arrival of several superb research associates.

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The first was Tsutomu Arakawa, who wanted to work with solvents. He started immediately on arrival and did not stop for the next five years. He was amazingly productive; he was a very careful laboratory scientist, who understood very well the theoretical underpinning of what he was doing and knew where to proceed. His devotion to work was amazing. One day, he came in with a high fever. M a r i n a Gorbunoff, with whom he shared a laboratory, ordered him to go home and not to appear until he was well. He obeyed. W h e n he left my laboratory, he went to work at Amgen where he applied his understanding of solvent effects to the folding of newly synthesized proteins and had a major influence on the early success of that company. There is an anecdote that goes along with this. One day I was giving a seminar at Amgen's competitor. Arakawa had applied there and had been t u r n e d down because of "difficulty of communicating" (he had some difficulties with English, which never caused a problem in our discussions). Everyone at the institution I talked with on that day would pop the question: "Tell me, what is Arakawa up to now?" My answer was: "Pick up the phone and ask him directly' They did regret their decision! Arakawa was the author of 21 publications from my laboratory. He explained a number of important phenomena in terms of preferential exclusion (reviewed in Ref. 61). First, he showed that the precipitation of proteins by salts (salting out) is modulated by the preferential exclusion of salts which led to the exact thermodynamic definition of the long used empirical "solubility constant"A detailed analysis of series of salts showed that weak ion binding could compete with the general preferential exclusion. This led to the formulation of the concept that the measured preferential exclusion is a balance between exclusion of the cosolvent (e.g., salt) due to some general force (perturbation of the surface tension, solvophobicity, or excluded volume) and actual binding by, e.g., hydrophobic interactions or electrostatic attraction. This was expressed by the relation vL(measured)=vL(eXclusion)+vL(binding), the first term on

470

S. N.TIMASHEFF

~

Co-Solvent

~f'~N~"-Volumeof Exclusion= Vs I

Fig. 5. Schematic representation of the steric exclusion of a cosolvent (e.g., PEG) from the surface of a protein. The volume of exclusion becomes occupied by the smaller water molecules; this manifests itself as preferential hydration (Rp = radius of protein; Re = radius of the excluded cosolvent). (Reprinted from Arakawa and Timasheff, Biochemistry, 1985.)

the right-hand side being always negative, the second always positive [62]. These were logically extended to the examination of the salting-out properties of the widely used protein crytallizers, polyethylene glycols (PEGs). All displayed preferential hydration which increased monotonely with the PEG molecular weight, meaning that PEG exclusion is caused by their excluded volume [63], a possibility already suggested by Lee and Lee [64]. This means (see Figure 5) that PEG molecules, because of their bulk, cannot approach a protein molecule by a distance smaller than their effective radius (they bump physically into the protein). That gap is filled with water which, in binding measurements, manifests itself as preferential hydration. Finally, Arakawa became interested in osmolytes. These are small molecules, usually sugars, polyols, amino acids, amino acid derivatives, or methylamines, which a variety of plants and animal vertebrate and invertebrate systems use to maintain osmotic pressure and osmotic flow. Now, all of these are known as "compatible solutes:' because they do not disturb cellular structure and function. Preferential interaction measurements showed them all to be preferentially excluded [65]. Why should nature use compounds that lead to preferential hydration? The very simple explanation is that, being preferentially excluded,

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these molecules do not come into contact with the surfaces of organelles w i t h i n the cell and, hence, c a n n o t d a m a g e them. This is t h e cause of their "compatibilityf. A r a k a w a left my laboratory in 1982, b u t the a m a s s e d results fed the literature for close to a decade, because of my slow process of performing in-depth analyses. The solvent-interaction work e a r n e d me an invitation to the m e e t i n g of the Cryobiology Society in South Carolina in 1991. This led to my discovery t h a t "honorary" is not limited to doctorate, b u t actually applies also to postdoctorate. So, on one hot s u m m e r day, I found myself in the c o m p a n y of a group of superb scientists; all were cell biochemists: George Somero, J o h n a n d Lois Crowe a n d their student, J o h n Carpenter. George Somero's brilliant work served as a catalyst in my f u r t h e r analyses of osmolyte t h e r m o d y n a m i c s a n d the role of osmotic pressure in biological function. J o h n C a r p e n t e r invited me to look at his poster in which his observations on the action of osmolytes found an explanation in preferential exclusion. While reading Arakawa's papers out of general intellectual curiosity, he h a d come to the realization t h a t this was the basis of the selection of osmolytes by nature. At t h a t time, a variety of explanations were offered for various types of molecules. This concept, however, was universal for all. Since then, J o h n C a r p e n t e r has become the minister of p r o p a g a n d a of preferential exclusion. He stresses t h a t our findings are the u n d e r p i n n i n g of how to fold newly synthesized proteins; he teaches this to e n g i n e e r i n g students a n d at workshops on protein folding a n d stability, while p u r s u i n g his o w n research p r o g r a m on p r o t e i n stability based on solid physico-chemical foundations. Even t h o u g h he has never been in my laboratory, I like to refer to h i m as my "postdoc honoris causa"

I n t e r a c t i o n of T u b u l i n w i t h A n t i m i t o t i c D r u g s F r o m the very inception of our work w i t h tubulin, one of the questions t h a t i n t r i g u e d us was how do certain drugs - namely,

472

S.N. TIMASHEFF

the vinca alkaloids (vinblastine and vincristine) and colchicine bind to tubulin and destroy microtubules (as observed in vivo) when introduced at very low concentrations, i.e., by a substoichiometric mechanism? We set out to examine this problem by a characterization of the binding of these drugs to tubulin and its linkage with the self-association properties of the protein. The vinca drugs were studied primarily by George Na and V. Prakash. The colchicine interactions were the domain of Jose Manuel Andreu. George Na and V. Prakash had absolutely contrasting personalities each leading in its own way to success in research. George Na was very reserved and independent. He insisted on learning everything on his own. He chose to work on the interaction of tubulin with vinblastine (VB) and came out with a solid study [66]. Prakash was just the opposite; he always welcomed suggestions. In the laboratory he was an automatic man; he planned his day to the minute, and invariably accomplished the plan. His productivity was enormous. His strong point was laboratory technique and he had an amazing instinct for the next experiment to do. When he arrived, we were starting experiments on the preferential interaction of 8 M urea with a series of denatured proteins [67]. There were problems of reproducibility. Prakash, who had never done a preferential binding experiment, joined the effort. In rapid order he identified the source of the difficulties. Aqueous urea solutions were leaching minute quantities of something out of the dialysis tubing. With great energy he devised protocols both for the purification of the commercial "ultra pure" urea and for the washing of the dialysis tubing before use. From that point on, all the results were reproducible. Prakash stayed for three years. He came back later for several short stays, during which he initiated the work on urea and guanidine HC1 denaturation. One of these visits came at a time when the chain of technical expertise in my laboratory had broken; the new postdocs had nobody to teach them the detailed procedures at the bench. In quick order, he taught them how to prepare pure tubulin in high yield and how

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to do preferential interaction measurements. His productivity and enthusiasm never waned and his massive data always remained reliable. At the time he was a research scientist at the Central Food Technological Research Institute in Mysore. Now he is the Director of that Institute. Jose Manuel Andreu was a mature scientist from Madrid. He understood rapidly what were the important questions to ask and pursued them systematically. This led him to a detailed characterization, using thermodynamic techniques, of the binding mechanism of colchicine on the submolecular level and of its consequences, namely substoichiometric inhibition of microtubules, tubulin self-association, and generation of GTPase activity. He had come in 1978 as a Fogarty Fellow; he is now a Professor in Madrid. Even though he left my laboratory in 1981, our active collaboration has continued even until and after my retirement. On the surface the vinca and colchicine systems would appear similar. Both inhibit microtubules substoichiometrically. Both are complex molecules, with two binding moieties. Vinblastine (VB) and vincristine (VC) consist of two submolecules joined by single bond; colchicine (COL) is a three.fused ring molecule (Figure 6). There are major differences in their interactions with tubulin. I shall not go into the details of the studies, but I shall just summarize the reasoning and logical development. Tubulin binds reversibly two molecules of VB [68] (VC), one of which induces a conformational change in tubulin which leads to self-association in a linear progressive isodesmic mode, thus precluding microtubule formation [69]. Colchicine (COL) binding induces a conformational change in tubulin which generates a GTPase activity not linked to polymerization (that associated with microtubule formation is). A second consequence of COL binding is the formation of polymers that mimic microtubules (need of GTP, inhibition by GDP, Ca 2+, and low temperature), but cannot fold into the cylindrical structure [70].The complex structures of the drugs raise the question of the role of the various structural elements of these molecules

474

S. N. TIMASHEFF HNCOCH3

HNCOCH 3

o COCH3 OCH3 CH3O-

T

~ OCH3

OCH3

Colchicine

CH30 OCH3

AIIocolchinone

Fig. 6. Structures of colchicine and the rationally designed analog, allocolchinone. In this analog the seven-membered tropone ring C of COL has been replaced with a six-membered phenyl ring (C'). The side chain on ring C' is a methyl ketone which, on binding to tubulin, imparts to the ligand a very strong microtubule inhibition capacity. (When the side chain is a methyl ester, as in allocolchicine, the inhibition capacity is diminished.)

in the interaction with tubulin: Jose Andreu started by a study of the individual thermodynamic contributions of the two end rings, A and C to the binding. By pushing the sensitivity of binding techniques, he was able to measure the free energies of binding of rings A and C individually [71]. Their sum was insufficient to account for the strong binding of COL. That deficiency in AG ~ ( C O L - (A + C)) turned out to be equal to the cratic free energy, that is the gain of the unfavorable entropy of mixing (cratic entropy) of one particle when fused into the three-ring structure (two particles become immobilized when A and C bind independently to the protein, whereas only one when they are fused, as in A-C, or A-B-C). This gives an additional binding strength of AGcrat-~-2.5 kcal/ mol. In fact, this is the same phenomenon as in Ron Frigon's analysis of the closing of the tubulin double ring. A systematic approach uncovered the contribution of each structural moiety to function: the bidentate binding occurs to independent noncooperative subsites, A and C, on the protein [72]. This is shown in Figure 7. All the functional properties reside in ring C: it binds first and induces in tubulin a conformational change that imparts to the protein all the new properties and brings

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C

)

1

Protein A

AI

]

Fig. 7. Schematic representation of the binding of a bidentate ligand, such as vincristine or colchicine, represented for colchicine binding. In the first step, ring C binds to its subsite on tubulin. This induces a conformational change in the protein (step 2), which brings into proper position the ring A binding subsite. In the third step, ring A falls into its subsite and locks the drug in position on the tubulin.

into proper spacial position the ring A binding site; ring A then folds into its pocket and serves as a lock on the stable complex. The role of ring B is passive [73]: it is only entropic in that it imparts rigidity on the COL molecule and precludes the unfavorable loss of the entropy of rotation between rings A and C when these bind productively to the protein. The contribution of each particular chemical group on the rings was approached by collaboration between Jose Andreu and Marina Gorbunoff, who synthesized a vast array of carefully thought out single ring, two-ring, and three-ring analogs of COL with systematic elimination of particular groups or their replacement by others. This mapped the thermodynamic contribution of each chemical feature of the colchicine molecule to the strength of binding and of its effect on tubulin function [74,75]. The culmination of this effort was the rational design and synthesis of a "super-colchicine:' which we called allo-colchinone [76] (see Fig. 6). It binds to tubulin strongly and reversibly, and is a reversible inhibitor of microtubules, stronger than COL, which renders it a better antimitotic agent. Following similar

476

s.N. TIMASHEFF

reasoning, Prakash demonstrated that the same bidentate mechanism governs the binding of the vinca alkaloids [77]. Again only one moiety carries the ability to induce changes in the properties of tubulin, the other one serving as an anchor. The molecular consequences, however, are different from those induced by COL. The uncovered thermodynamic linkages in the interactions of tubulin with various ligands aroused our curiosity about geometric ones. This was examined in measurements of distances between sites; Octavio Monasterio did this by relaxation n m r [78], while L a r r y Ward and Robert Seckler approached this by nonradiative fluorescence energy transfer [79]. In this way we obtained a general structural picture of the linkages that exist in the ~-fl tubulin dimer. A number of these allosteric actions occur over large distances.

The Final Years Tubulin behavior is controlled by the curved <-->straight isomerization. It all comes together, as tubulin assembly, drug binding, and solvent-interactions coalesce After close to a ten-year lapse, work on tubulin self-associations resumed. This was prompted by arguments in the literature on the role of nucleotide exchange in tubulin. The conclusion reached by Robley Williams, Jr. and his laboratory [80] that tubulin seemed to exist in two conformations and their careful measurements of Mg 2+ binding to tubulin liganded to GDP and GTP induced us to examine the self-associations of tubulin with GDP in the exchangeable site. A sedimentation velocity examination of double-ring formation of GDP-tubulin revealed that tubulin liganded to GDP forms these structures very strongly, much more so t h a n GTP-tubulin [81]. The data could be analyzed only if one invoked an equilibrium between two species, which differed only in the mutual geometric location of the polymerization sites at the two poles of the a-fi

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dimer. In the presence of GDP, tubulin exists almost exclusively in the ring-forming (curved) conformation (it forms rings but is incapable to form microtubules); GTP-tubulin, on the other hand, exists in an equilibrium between curved and straight conformations. The latter assemble to microtubules, but double rings are also always seen in the electronmicroscopic pictures [53,81]. The postulation of the curved ~->straight equilibrium opened a vista on the numerous observations of ~curled structures" in microtubule studies. A concerted molecular and thermodynamic picture could be obtained by sophisticated kinetic experiments. And so, 1986-87 found me in Gif-sur-Yvette on a sabbatical with Dominique Pantaloni and his wife, MarieFrance Carlier, two very keen intellects. My first contact with Dominique Pantaloni had been in 1958 when he was a graduate student in Tonelat's laboratory. His thesis project was on fl-lactoglobulin, the protein which at the time was central to my interests. Thirty years later, our interests coalesced again. Dominique had become interested in the process of microtubule assembly and function. Our approaches were different, but complementary. Dominique understands thermodynamics profoundly. Marie-France, endowed with a very quick, brilliant mind, can t h i n k up and carry out highly original and intricate kinetic experiments that go to the core of an issue. Collaboration with them and their talented student, Ronald Melki, produced definite proof of the GTP-GDP controlled straight ~-~curved tubulin conformation equilibrium which determined whether tubulin will self-assemble into microtubules or double rings, keeping the intermolecular contacts identical [82] (shown schematically in Figure 8). It also established the molecular pathway of microtubule disassembly with the eventual formation of double rings. The question to answer at this point was what are the switches that control this conformational equilibrium? This question was unraveled by Keith Shearwin, who joined my laboratory in 1990. That year, in fact, saw the arrival of my last postdoctorals. A greater set of contrasting personalities is h a r d to imagine. Yet, each was talented in his own way, and

478

S. N. T I M A S H E F F

A. TubulinConformations "Straight. L

.

.

.

Curved" 27~L

L

L

B_AssemblyModesof "Straight"and "Curved"Tubulin

.4

/" /'7

L U"7

(:': I

Fig. 8. The curved ~-~straight tubulin equilibrium. Part A depicts schematically the two conformations. With Mg-GTP as ligand, the protein exists predominantly in a structure in which the two microtubuleprotofilamentgrowth sites (L) are in linear arrangement. With GDP as ligand, a bend occurs at the interface between the ~ and fl subunits 4 tubulin, so that polymerization can occur only in a curved configuration. Part B shows a growing microtubule; incorporation of a curved isomer of tubulin causes the protofilament to bend away from the axis of the mierotubule which stops microtubule growth; further additions initiate the formation of the closed-ring polymer. (Reprinted from Melki et al., Biochemistry, 1989.)

helped to create the last "golden years" of my laboratory. Keith was a thoroughly trained physical-chemist from Don Winzor's laboratory in Brisbane. He was able to undertake protein thermodynamic analysis on the highest level and to develop complex

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linkage schemes and thermodynamic boxes, exactly what was needed at this point. His experimental tool was almost exclusively the old reliable Model E ultracentrifuge, in its two modes, sedimentation velocity and sedimentation equilibrium in which he pushed the analysis with the UVabsorption scanner to the lowest possible limit of protein concentration. By a systematic manipulation of nucleotides, divalent cations, colchicine, and dissociation of tubulin into ~ and fl subunits at extremely low protein concentration, Keith found that, in the absence of ligands other t h a n nucleotides, both GTP and GDP tubulins exist in the curved state and form double rings with identical free energy [83]. This established that "curved tubulin" is the ground,

relaxed, state of tubulin, all others being at higher free-energy levels, confirming quantitively the intuitive conclusion reached by Dick Weisenberg in 1976 that double rings are the storage form of tubulin [47]. Analysis over a series of linked thermodynamic boxes demonstrated that the conversion of ground state "curved tubulin" to the "straight" microtubule forming isomer is a very unfavorable reaction; it occurs with an equilibrium constant of < 0.01. This blocking is lifted by binding to the y-phosphate of the exchangeable tubulin GTP of a Mg 2+ ion with a high affinity (Kb = 106M-1). It is this Wyman-type linkage of the strong ion binding to the very weak isomerization which leads to the requirement of a high Mg 2+ concentration in microtubule assembly [83]. W h e n Keith left, the problem of the allosteric controls of microtubule assembly had been essentially solved. Bernardo Perez-Ramirez arrived shortly after Keith. Bernardo had been advised by his PhD mentor that he was grossly unprepared for the kind of studies that we did, a message that I was also given, and a fact that he fully acknowledged. He came to learn, and learn he did! He shared a laboratory with Keith. Their interaction was symbiotic: as much as Keith was systematic, so much was Bernardo impulsive. Keith was strong in thermodynamics and Bernardo's strength was biochemical techniques; both were extremely careful experimentalists and got

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along splendidly. They collaborated extensively, sharing their individual expertises. This is the ideal that should exist in all laboratories. Bernardo became interested in the colchicine system and, in particular, in the induction of a weak GTPase activity by the drug. Careful comparison of the binding equilibrium measured by optical techniques and the generation of enzyme activity led to the discovery of a postbinding thermodynamically unfavorable conformational transition which induces the GTPase activity in the protein [84]. Preferentially excluded cosolvents were found to enhance the activating transition without affecting the binding step. Proper manipulation of cosolvents demonstrated that, intrinsically, the tubulin-COL GTPase activity is strong; its observed weakness simply reflects linkage to the unfavorable equilibrium between the liganded inactive and active forms of the protein; in dilute buffer, less than 10% of the protein is in the active form. Tiao-Yin Lin and Guifu Xie, both of whom also arrived at that time, were excellent experimentalists. Both went to work on new aspects of protein-solvent interactions which crossed new horizons. Tiao-Yin characterized two thermodynamic compensations. The first was between thermal denaturation and preferential exclusion [85]. Her demonstration that, for sugars, the thermal unfolding takes place at identical values of solvent surface tension, means that unfolding has to overcome an invariant free-energy barrier. The other compensation gave a thermodynamic explanation for a biological fact. It is known that certain sharks accumulate urea as osmolyte, which they compensate with methylamines [86]. Urea is a denaturant and methylamines are stabilizers. Doing preferential interaction measurements in four-component systems (water-protein-urea-methylamine), Tiao-Yin showed that the urea-methylamine levels found in the fish corresponded to points at which the preferential binding of urea was compensated exactly by the preferential hydration induced by the methylamines [87]. The net effect was a zero free energy of protein-solvent interaction, and no effects of the osmolytes on physiological structure. This led us to the

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Third Law of Molecular Sociology: "Fishes and frogs, but also plants and invertebrates, obey molecular thermodynamics in everyday life much more diligently than does homo sapiens!" Guifu Xie t o o k on detailed studies of t h e interactions of protein w i t h a sugar (trehalose) [88] a n d polyol (sorbitol) (reviewed in Ref. 61), b o t h s t r u c t u r e stabilizers, and with u r e a [89] a n d g u a n i d i n e hydrochloride. Concerted experiments of b i n d i n g a n d p r o t e i n d e n a t u r a t i o n led to a detailed understanding of the interactions. She was successful in m e a s u r i n g independently the interactions of the stabilizers w i t h the protein in the native a n d d e n a t u r e d states. The differences m a t c h e d exactly those deduced by Wyman analysis from equilibrium unfolding measurements. She t h e n u n d e r t o o k a study of the effect of t e m p e r a t u r e on preferential interactions. To my knowledge, she is the only one to have succeeded in this; she obviously did not recognize the difficulty involved. Her laboratory technique was so good t h a t she almost routinely overcame difficulties t h a t h a d s t u m p t e d m a n y others. Her studies gave the thermodynamic p a r a m e t e r s of the preferential interactions w i t h proteins of b o t h stabilizers a n d denaturants. Now in my retirement, her data still occupy me w i t h in-depth analyses of interactions, an intellectually s t i m u l a t i n g exercise.

Retirement, Epilogue Sometime in 1994, I applied for my routine NIH g r a n t renewal, a process in which I h a d always come n e a r the top. This time, Fate o r d a i n e d differently. My score came out low on the list. The c o m m e n t was "In the 1990s one does not measure binding constants" a n d I was i n s t r u c t e d to clone dozens of m u t a n t s of t u b u l i n a n d see how they behave. In one sentence, Wyman, m u l t i c o m p o n e n t t h e r m o d y n a m i c s a n d complex linkages were dismissed as trivial m e a s u r e m e n t s of binding constants. I closed my laboratory a n d retired: sic t r a n s i t Gloria mundi! After an initial b u r s t of anger, I realized t h a t I was free. I was

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free to t h i n k u n d i s t u r b e d about problems for which I seemed never to have h a d the time. There were no deadlines, no grants, no teaching, no administration. Now, I could write didactic papers on protein-solvent interactions. These explained the subtle differences which exist between p r o t e i n hydration a n d preferential hydration, two f u n d a m e n t a l l y simple, b u t independent, t h e r m o d y n a m i c concepts, but the confusion of which h a d led to the appearance in the literature of u n f o r t u n a t e misconceptions [90]. I decided to explain the equations with simple pictures t h a t rendered the abstract visible. This p u r s u i t was paralleled by a friendly scholarly correspondence w i t h J o h n S c h e l l m a n w h o m I had k n o w n as an o u t s t a n d i n g scholar in p r o t e i n conformation, denaturation, a n d spectroscopy. It is truly an anecdote t h a t J o h n a n d I were b o t h working for years on protein-solvent interactions, totally oblivious of each other's activity. J o h n was developing the theory, while my laboratory was doing the measurements. A n almost accidental e n c o u n t e r revealed our m u t u a l interest. John's theoretical development took protein-solvent interactions to new frontiers. It is his work t h a t solved for me a great puzzle: how could positive b i n d i n g c o n s t a n t s lead to negative b i n d i n g m e a s u r e d in dialysis equilibrium experiments? At this point we were c o m p a r i n g our u n d e r s t a n d i n g of some f u n d a m e n t a l aspects of what was being measured. To my delight, we found ourselves in full a g r e e m e n t not only on what we know a n d u n d e r s t a n d , but also on some of the questions t h a t need to be answered. It is such collaboration in total collegiality t h a t makes science an enjoyable a n d worthwhile pursuit. W h a t a pity it is to see nowadays the so frequent rivalries a n d races to publication. L o o k i n g back at my career, I derive great satisfaction in having i n t r o d u c e d a n u m b e r of young scientists to the power of W y m a n linkages. All my years at Brandeis I have t a u g h t the physical biochemistry course. My aim was to i m p a r t a solid basis on which biochemists could develop quantitative schemes of biological controls. A large p a r t was devoted to interactions

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and a detailed presentation of Wyman linkages a n d related thermodynamics. The presentation was systematic a n d analytical a n d s t a r t e d from first principles. It was abstract and, hence was detested by the students. A n d yet some of the former students would find it necessary some t e n years later to express to me their gratitude for having t a u g h t t h e m the Wyman theory. They found themselves far a h e a d of their new colleagues w h e n it came to unraveling biological controls. Molecular biology colleagues in the D e p a r t m e n t tried to dissuade me from teaching abstract theory. One year, w h e n I was going away on sabbatical, the course was t a u g h t by a m u c h younger faculty member, who u n d e r s t a n d s physical chemistry in-depth. In looking over my course outline he asked me: "Why do you teach all the Wyman stuff?." I just said: "Why don't you read it in the original?" a n d gave h i m references. He obviously did read it, for sometime later he came to me a n d showed me how application of Wyman's t h e o r y h a d helped h i m solve a problem in ion transport. He was not the only one to w h o m Wyman h a d opened a new world. Not only did he teach it t h a t year, in a presentation different from mine, but, now t h a t I am retired, W y m a n linkages are solidly i m p l a n t e d into the c u r r i c u l u m of t h a t course at Brandeis. In fact, he t u r n e d out to be the only colleague at Brandeis with w h o m I could t h r a s h out questions of solution thermodynamics; otherwise, I was on my own. In t a l k i n g about my laboratory, I have m e n t i o n e d specifically only some very good postdoctorals. There were also a n u m b e r of less talented ones, some mediocre. With those who were trying, I spent m u c h more time t h a n with the good ones, helping t h e m to leave w i t h their n a m e s on at least one paper. A few were total failures. There was one, however, w h o m I caught cheating, fortunately before any h a r m h a d been done to my program. He did not stay long. In fact, he was a very nasty fellow. At the time, there was a R u m a n i a n - J e w i s h t e c h n i c i a n in t h e laboratory, a refugee whose family h a d suffered from the Ceausescu government. He would repeatedly s t a n d next to her a n d whistle the

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"Internationale." This practice was dealt with abruptly in front of the whole group. Otherwise, t h e atmosphere was friendly a n d the postdocs would as a rule help each other. There were some rivalries. One, b o r d e r i n g on the childish, was between two very good ones who were c o m p e t i n g as to who could get a higher yield of very pure t u b u l i n in a prep. This did not interfere with progress, although they stopped t a l k i n g to each other. The benefit was a c o n s t a n t improvement in the tublulin isolation procedure. Not only did r e t i r e m e n t free me to p u r s u e science intellectually, without any pressure, it also gave me the freedom to move as I wanted. We h a d always enjoyed traveling, a n d now we were no longer constrained. We discovered the magic beauty of the n o r t h e r n sky on a boat trip along the N o r w e g i a n coast, the spectacular a n d savage beauty of the tip of S o u t h America, glaciers coming down into the ocean in the Beagle Channel, the wilderness of Torre del Paine, Lago A r g e n t i n a a n d the savage gusts of w i n d t h a t sweep the P a m p a from one end to the other. We gazed at Iguazu Falls with its t h o u s a n d s of fabulous butterflies a n d most u n u s u a l fauna. We became acquainted w i t h the myriads of birds on the Galapagos Islands. We saw Machu-Picchu a n d crossed the Andes by t r a i n through, as it became clear on the spot, a region infested by the Sendero Luminoso at t h e height of its activity. We h a d already seen India, Japan, Egypt, the Holy Land, the desert of S o u t h e r n Morocco and h a d learned how to haggle in the souks of Marrakech. We had seen the Maya ruins of Guatemela and Honduras, driving a jeep between the two. The "Fall of the Berlin Wall" opened new vistas to us, a m o n g the most e n r i c h i n g ones being Prague, Budapest, a n d w h a t r e m a i n s of the baroque jewel Dresden, so savagely destroyed in the last days of World War II. Finally, at the millennium, we went on a trip to Russia a n d I set foot in the c o u n t r y of my ancestors three-quarters of a c e n t u r y after I was born. This was the s t a n d a r d boat trip from St. Petersburg to Moscow on the Golden Ring.

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After a three-day visit of the magnificent m u s e u m city of St. Petersburg, totally non-Russian, where b o t h my p a r e n t s were born, t h e boat set out on t h e Neva into lake Ladoga where it made its first stop on the island of Kiji, the m u s e u m of t h e r e n o w n e d n o r t h e r n Russian wooden churches. We were s t r u c k by their similarity to the stavkircher (wooden churches) of Norway. In this there was n o t h i n g surprising: the two are creations of the Vikings; one m u s t recall t h a t the Russian state (Rus) was founded by the Varingian prince, Rurik, in Kiev. The n o r t h e r n waterways were p a r t of their trade route to Constantinople, a n d Kiev was on the way. Following the canals a n d lakes we finally emerged on the Volga river, "Mother Volga:' the a r t e r y of Russia. Sailing on the quiet waters of the Volga, we looked at the peaceful a n d never c h a n g i n g banks, lined w i t h woods. This is so in h a r m o n y w i t h the melancholy somewhat apathetic spirit characteristic of Russians, the spirit of the neverending plain, the monotonous, yet so peaceful, river banks. This spirit is manifested in the slow, graceful village dances, t h a t now we see performed on stage. Every few kilometers, the dome, usually blue, of a c h u r c h would appear among the trees, a n d one would t h i n k of the Russia t h a t was: most of these domes are on a b a n d o n e d churches brought to r u i n by years of the Soviet regime. They are p a r t of t h e past, culturally now a very d i s t a n t past. They are history, almost ancient history. A n d this brings to m i n d t h e r e m a r k made to me by the young Soviet scientist: "You are our history?At this point I realized how m u c h this is true, for I have m u c h more kinship to these a b a n d o n e d blue domes t h a n to the people who emerged from u n d e r the Soviet yoke. To grasp their spirit, their mentality is as difficult a task for me as it is for t h e m to reach to me as "their h i s t o r ~ A t times, one sees on the b a n k some a b a n d o n e d factory: this is a testimonial to the n a t u r a l collapse of a nonviable system, while the r u i n e d churches are a symbol of an i m p o s e d destruction of a culture t h a t h a d flourished for one t h o u s a n d years. Gliding along the water, late in my life, I finally was seeing "Holy Russia:' the old cities of Iaroslavl, Kostrama, Uglitch w i t h

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their magnificent churches, the Kirilo-Belozerski monastery. We managed private side trips to the Pherapont Monastery covered with frescoes by the great Dionisii, and the Holy TrinitySt. Sergei Monastery which houses the highly venerated relics of St. Sergei of Radonezh. In Moscow, we contemplated the Cathedral of the Dormition with its famous Roublev iconography. This was the Russia that had given birth to the culture in which I had grown up, in "Little Russia:' On walking around Moscow, my wife recognized the little green square where she had played as a little girl and the apartment house which she left with her mother forever almost 60 years before. Our French fellow tourists on the boat would ask me: is my reaction one of deep emotion? No, it was not. It was rather one of philosophical contemplation: this was all history, an era finished forever, to which these magnificent monuments stood as silent witnesses. They were testimonials to Russian and, in fact, Byzantine mysticism. The systematic destruction of this spirit over three-quarters of a century has culminated now in current Russians asking us for help in seeking their roots. The destruction was not only spiritual, but also moral and material. Heart rending was the sight of the poverty all around us and, most depressing, the sight of 14-year-old boys sitting d r u n k on the border of the canal in Iaroslavl. It took the span of three generations to do the destructive work, it will take as much to do the rebuilding. Today, what remains of the Russian emigration and some of their progeny, who mostly do not speak Russian, are doing what they can to help the myriads of destitute orphans [91]. The emphasis is on the children for they are the future of the nation. On the boat I became inadvertently an informal secondary guide and source of information to passengers. Among the various programs, there was a course in Russian. As the "students" would then practice their lesson, I would casually correct pronunciation. Soon, they discovered that one of what they called "the famous White Russians" was on board. I would be asked questions about Russian history, the Orthodox

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religion, icons, the significance of various features of the churches, etc. This was a touristic tour with a strong cultural overhang. Along with the visits to the sites, there was a daily lecture on aspects of Russian culture and a daily historical film, shown in six parts, called "From Lenin to Gorbache~' Using films from the recently opened Soviet archives which had recorded events from the start of the revolution, this documented the atrocities directed by Lenin (whom UNESCO had feted as a great humanitarian): his arrest of thousands of intellectuals in 1918 and their exile to concentration camps in the north; his brutal war against the peasants which caused the famine of 1920, etc. Most sickening were the pictures of the intellectuals digging the Volga-White sea canal, while a small orchestra played marches to the tune of which the slaves had to move their shovels. The hall in which this was shown was overfilled every day. Every morning, over the boat intercom connected to every cabin, there would be a wake-up call, followed by a reading of the day's program, with invariably: "Today we will sail along the 'XYZ' canal, the construction of which cost 5 raillion (7 million) lives" The freedom of retirement also gave the tranquility to t u r n to the roots of our culture. This is the Judeo-Christian culture amalgamated with the philosophy of Greece. This is the culture that for a millennium was centered in the Eastern Roman Empire, with its capital in Constantinople. This culture, in its transcendental spirit, had preserved ancient Greek philosophy and art and, in its late years, transmitted them to the West and, thereby, catalyzed the start of the Renaissance. The spirit of that culture, which permeated the entire Christian world, from Armenia to France, Spain, and the British Isles, can be fathomed in the Romanesque architecture of the 12th century which was a flourishing period. The perfect proportions of the churches at the monasteries of Senanque or Thoronet, which have no decorations and no sculpture, bring on a state of inner peace and forgetfulness of the turmoil in which we live (Gothic architecture does not have that power). The same is true of the

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s t a t u a r y of the same period. One only has to s t a n d in front of the elongated, almost abstract one-dimensional statues on t h e cathedral of Moissac. They are three-dimensional iconography; we have seen their c o u n t e r p a r t s as frescoes on t h e walls of the small 13th-14th century basilicas of the towns of Greek Macedonia a n d on the island of Patmos. (It is a tragedy t h a t the wonders of Kosovo, classified as World Treasures by UNESCO, have been u n d e r g o i n g systematic destruction over the past five years.) These frescoes, totally introspective, just as the Moissac statues, lead to m e d i t a t i o n a n d their contemplation induces a state of serenity, as intended. Iconography is not art; w h e t h e r two dimensional or t h r e e dimensional, it does not represent reality; it is a message to be read a n d understood. My direct e n c o u n t e r w i t h the 14th-15th c e n t u r y Russian iconography evoked a great interest in Byzantine art on which it is founded. A n d so, we followed the evolution of iconography from its artistic origins in the mosaics a n d portraits of ancient Rome, t h r o u g h the magnificent 5th-12th c e n t u r y mosaics of Ravenna a n d Thessaloniki, as well as those of Palermo. In Istanbul we have seen the little t h a t r e m a i n s of the wonder of Aghia Sophia (most of the mosaics were destroyed in 1204 by the F r a n k i s h crusaders d u r i n g the 4th Crusade), the magnificent mosaics a n d frescoes of Chora basilica a n d mosaics, still preserved in a few little k n o w n small basilicas, now mosques, to which we gained access by perseverance a n d a b a k s h e e s h to the right person. One of our pleasures is to travel to art exhibitions; it can be to Washington for Velasquez, Chicago for Caillebotte, or New York for E1 Greco, not to m e n t i o n Paris, London, A m s t e r d a m , etc. I have learned that frequently it is only at an exhibition, which amasses a large n u m b e r of works by the same artist, t h a t I get to u n d e r s t a n d him. While this is not n e e d e d for the Renaissance p a i n t i n g s of Raphael, which are beauty in its perfection, it is in this way that, for example, I realized the miracle performed by E1 Greco in m a r r y i n g the m o t i o n and turmoil of m a n n e r i s m with the contemplative iconographic expressions of the faces, the eyes, t h a t convey spiritual

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m e s s a g e s f a r b e y o n d t h e d e p i c t e d t h e m e . R e a d i n g a b o u t E1 G r e c o a f t e r t h e e x h i b i t i o n , I l e a r n e d t h a t t h i s f a c t is well k n o w n to the specialists. In general, my favorite paintings are p r e - R e n a i s s a n c e a n d t h e N o r t h e r n School, b u t also t h e b r i l l i a n t I m p r e s s i o n i s t s , w i t h t h e i r m e s s a g e of i n n e r joy a n d o p t i m i s m , i n d r a s t i c c o n t r a s t to t h e i r g l o o m y G e r m a n c o n t e m p o r a r i e s . R e t i r e m e n t is a d e l i g h t f u l a n d u p l i f t i n g p a r t of life. I do n o t i n t h e l e a s t r e g r e t t h a t t h e s t a t e of e m e r i t u s w a s f o r c e d u p o n me. I n fact, I a m t h a n k f u l for this.

REFERENCES [1] Much of what I shall tell comes from memory either of what I have witnessed myself or of what I have been told by members of the preceding generation. I ask for the indulgence of the readers, therefore, for any inaccuracies in details of my composition. [2] Denikin, A.I. (1922) The Russian Turmoil. NewYork, E. P. Dutton and Co. [3] Pipes, R. (1990) The Russian Revolution. NY, A. A. Knopfi [4] Lehovich, D.V. (1973) White Against Red. NY, W.W. Norton. [5] Katkov, G. (1967) Russia 1917. NY, Harper and Row. [6] Nord, F.F., Bier, M. and Timasheff, S.N. (1951) J. Am. Chem. Soc. 73, 289-293. [7] Brown, R.A., Shumaker, J.B., Jr., Timasheff, S.N. and Kirkwood, J.G. (1952) J. Am. Chem. Soc. 74, 460-462. [8] Kirkwood, J.G. and Goldberg, R.J. (1950) J. Chem. Phys. 18, 54-57. [9] Kirkwood, J.G. and Shumaker, J.B. (1952) Proc. Natl. Acad. Sci. USA 38, 863-871. [10] Timasheff, S.N., Dintzis, H.M., Kirkwood, J.G. and Coleman, B.D. (1957) J. Am. Chem. Soc. 79, 782-791. [11] My translation (I am not a poet) of the poem "A Cossack lullaby", [12] Tanford, C. and Kirkwood, J.G. (1957) J. Am. Chem. Soc. 79, 5333-5339; Tanford, C. (1957) J. Am. Chem. Soc. 79, 5340-5347. [13] Townend, R., Kiddy, C.A. and Timasheff, S.N. (1961) J. Am. Chem. Soc. 83,1419-1423. [14] Townend, R.,Winterbottom, R.J. and Timasheff, S.N. (1960) J. Am. Chem. Soc. 82, 3161-3168. [15] Gilbert, G.A. (1955) Discussions Faraday Soc. 20, 68-71. [16] Townend, R. and Timasheff, S.N. (1960) J. Am. Chem. Soc. 82, 3168-3174. [17] Aschaffenburg, R. and Drewry, J. (1955) Nature 176, 218-219.

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[18] Kumosinski, T.F. and Timasheff, S.N. (1966) J. Am. Chem. Soc. 88, 5635-5642. [19] Kalan, E.B., Gordon, W.G., Basch, J.J. and Townend, R. (1962) Arch. Biochem. Biophys. 96, 37~381. [20] Kiddy, C.A., Townend, R.E., Thatcher, WTW.and Timasheff, S.N. (1965) J. Dairy Res. 32, 209-217. [21] Kirkwood, J.G. and Mazur, J. (1952) Compt. Rend. 2~ Reunion Chim. Phys., Paris 143-146. [22] Timasheff, S.N. and Kronman, M.J. (1959) Arch. Biochem. Biophys. 83, 60-75. [23] Bernardi, G. and Griff6, M. (1964) Biochemistry 3, 1419-1426. [24] Colter, J.S. and Brown, R.A. (1956) Science 124,1077-1078. [25] Timasheff, S.N.,Witz, J. and Luzzati,V. (1961) Biophys. J. 1,525-537. [26] Timasheff, S.N. (1964) Biochim. Biophys. Acta 88, 630-641. [27] Witz, J., Timasheff, S.N. and Luzzati, V. (1964) J. Am. Chem. Soc. 86, 168-173. [28] Timasheff, S.N. (1963) Electromagnetic Scattering (Kerker, M., ed.), pp. 337-355. NewYork, Pergamon Press. [29] Noelken, M.E. andTimasheff, S.N. (1967) J. Biol. Chem. 242, 5080-5085. [30] Timasheff, S.N. and Inoue, H. (1968) Biochemistry 7, 25014513. [31] Inoue, H. and Timasheff, S.N. (1972) Biopolymers 11,737-743. [32] Pittz, E.P. and Timasheff, S.N. (1978) Biochemistry 17, 615-623. [33] Wyman, J., Jr. (1948) Adv. Protein Chem. 4, 407-531. [34] Wyman, J., Jr. (1964) Adv. Protein Chem. 19, 223-286. [35] Tanford, C. (1969) J. Mol. Biol. 39, 539-544. [36] Hallerbach, B. and Hinz, H.-J. (1999) Biophys. Chem. 76, 219~27. [37] Aune, K.C. and Tanford, C. (1969) Biochemistry 8, 45794586. [38] Aune, K.C. and Timasheff, S.N. (1971) Biochemistry 10, 1609-1617. [39] Aune, K.C., Goldsmith, L.C. and Timasheff, S.N. (1971) Biochemistry 10, 1617-1622. [40] Wright, H.T., Kraut, J. and Wilcox, P.E. (1968) J. Mol. Biol. 37, 363-366. [41] Gorbunoff, M.J. (1971) Biochemistry 10, 250-257. [42] Lee, J.C. and Timasheff, S.N. (1974) Biochemistry 13, 257-265. [43] Hade, E.P.K. and Tanford, C. (1967) J. Am. Chem. Soc. 89, 5034-5040. [44] Weisenberg, R., Borisy, G.G. and Taylor, E.W. (1968) Biochemistry 7, 4466-4479. [45] Weisenberg, R.C. andTimasheff, S.N. (1970) Biochemistry 9, 4110-4116. [46] Weisenberg, R.C. (1972) Science 177,1104-1105. [47] Weisenberg, R.C., Deery, W.J. and Dickinson, P.J. (1976) Biochemistry 15, 42484254. [48] Frigon, R.P. and Timasheff, S.N. (1975) Biochemistry 14, 45594566. [49] Frigon, R.P. and Timasheff, S.N. (1975) Biochemistry 14, 4567-4573.

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[80] Croom, H.B., Correia, J.J., Baty, L.T. and Williams, R.C., Jr. (1985) Biochemistry 24, 768-775. [81] Howard, W.D. and Timasheff, S.N. (1986) Biochemistry 25, 8292-8300. [82] Melki, R., Carlier, M.-F., Pantaloni, D. and Timasheff, S.N. (1989) Biochemistry 28, 9143-9152. [83] Shearwin, K.E., Perez-Ramirez, B. and Timasheff, S.N. (1994) Biochemistry 33, 885-893. [84] Perez-Ramirez, B. and Timasheff, S.N. (1994) Biochemistry 33, 626~6267. [85] Lin, T.-Y. andTimasheff, S.N. (1996) Protein Science 5, 372-381. [86] Yancey, P.H. and Somero, G.N. (1979) Biochem. J. 183, 317-323. [87] Lin, T.-Y. and Timasheff, S.N. (1994) Biochemistry 33, 12695-12701. [88] Xie, G. and Timasheff, S.N. (1997) Biophysical Chemistry 64, 25~ 43. [89] Timasheff, S.N. and Xie, G. (2003) Biophysical Chemistry 105, 421-448. [90] Timasheff, S.N. (2002) Biochemistry 41, 13473-13482. [91] For example, through the Russian Children's Welfare Society, located in New York, which supports many orphanages and children's medical facilities in Russia.