Protein crystal growth rates determined by time lapse microphotography

Journal of Crystal Growth 110 (1991) 177-181 North-Holland

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Protein crystal growth rates determined by time lapse microphotography Stanley Koszelak, David Martin, Joseph Ng and Alexander McPherson * Department of Btoehemtstry. Umverstty of Cahforma at Rwer~tde, Rwerstde, Cahforma 92521, USA

Time lapse wdeo microscopy has been used to make quahtatlve observations of the events that transpire during normal and abnormal protein crystal growth It has also been used to make quanutatlve assessments of growth rates for a variety of different protein crystals From analyses of the growth rates, we have estimated that m the most rapidly growing crystals we have recorded, as many as 20 layers of protein molecules add to a single crystal face per second In the slowest cases of growth, such as virus crystals, a minute or more may be required for addition of a single layer In almost all cases, growth was hnear over nearly the entire period of growth before levehng near growth termination We present here a small but typical sample of the results obtained using the t~me lapse video microscopy techmque.

1. Introduction M a n y p h e n o m e n o l o g i c a l p r o b l e m s can be solved or at least u n d e r s t o o d w h e n their details are simply subjected to careful scrutiny. Often this requires n o more t h a n very careful o b s e r v a t i o n of the series of events that comprise the process. H o w a m m a l s move their legs in r u n m n g or liquid drops splatter, for example, b e c a m e clear only with the a d v e n t of high speed p h o t o g r a p h y [1]. Similarly, slow processes such as cell division or organ d e v e l o p m e n t were u n d e r s t o o d when time lapse sequences allowed the m i n d to integrate a vast series of small events a n d m o v e m e n t s i n t o a coherent process. W e beheve that p h o t o g r a p h y of crystals, particularly time lapse p h o t o m i c r o s c o p y of growmg crystals, m a y provide a useful tool for elucidating the m e c h a n i s m s a n d kinetics of macromolecular crystal growth, a n d could suggest factors that c o n t r i b u t e to the f o r m a t i o n of defects a n d imperfections. A t the Second I n t e r n a t i o n a l C o n f e r e n c e o n Macromolecular Crystal G r o w t h , we described an mexpensive a p p a r a t u s for recording time lapse * To whom all correspondence should be addressed

sequences of growing p r o t e i n crystals, a n d reported some qualitative observations regarding the growth process [2]. Since that time, we have extended our studies to a n u m b e r of other proteins a n d also to an icosahedral virus k n o w n as satelhte tobacco mosaic virus (STMV). U s i n g the same microscope a n d video system to record the images, we have e n d e a v o r e d to q u a n t i t a t e certain features of the growth process a n d translate these into physical m e c h a n i s m s . O n e of the simplest a n d most straightforward k i n d s of m e a s u r e m e n t s we can m a k e is of crystal growth rates. These c a n be m a d e b y simply c a l i b r a t i n g video screen d i m e n s i o n s to actual physical d i m e n s i o n s , m e a s u r i n g directly from the screen the lengths of crystal edges as a f u n c t i o n of time, a n d c a l c u l a t m g growth rates. Because we k n o w the d i m e n s i o n s of the p r o t e i n or virus particles f o r m i n g the crystals, we c a n estimate in a straightforward m a n n e r the rate of a d d i t i o n of i n d i v i d u a l molecules, or layers of molecules, to the growing crystal surfaces. The few cases that we present below represent only a small p o r t i o n of the m a n y h u n d r e d s of crystal growth sequences that we have recorded a n d are still analyzing. They do, however, 11-

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S Kos:elak et al / Protem cr~ ~tal ,~rowth rates deterrnmed by ttrne lapse mwrophotographv

lustrate the use of the method and the range of values for the growth rates that we have observed. In the most dramatic cases, we feel the growth rates are substantially greater than previously thought and certainly of such magnitude that mass transport processes may play an important role. It is difficult to describe in words vast numbers of visual observations so the reader must be patient with our discussion. Most of the observations and data described in tins paper have, however, been assembled on a single video tape. This tape is available to interested parties upon request from the authors.

2. Materials and methods

Crystalhzation experiments utilizing the v i n o u s proteins were carried out by the vapor diffusion method [3] using, primarily, sandwich drops in winch the 25 to 30 /~1 protein crystallization samples were maintained between two closely spaced glass slides (the drop being in contact with both) whale exposed as well to a reservoir solution through the vapor phase. The apparatus used was the ACA crystallization plate manufactured by F L O W Labs. The major advantage of tins procedure was the superior optics afforded by the slide-solution-slide arrangement. In other cases, more traditional hanging drop experiments were employed using 20 /~1 protein solution samples suspended from sihconazed 22 mm cover slips. The reservoirs were supplied by Linbro 24 well tissue culture plates, also manufactured by F L O W Labs The crystallization plate was positioned on the stage of a Swift microscope (model ~ M 4000-D) equipped with 4 × and 10 × objectives, crossed polarizer/analyzer, heat filter, and interfaced through a prism to a Sanyo color video camera (model # V D C 3900). Images were observed in real time on a Sony video monitor (model ~ KV 1396R) and recorded on a G Y Y R time lapse VHS VCR (model ~ T L C 1400). The entire system may be obtained in fully integrated form from Resolution Technologies, San Juan Capistrano, California. This unit has the ablhty to record images from real time to a compression of 120-fold. Most observations in tins study were recorded at a time

compression of 60-fold. Final magnification of the linage depends, of course, on the monitor being used, but a conversion factor derived from observing a stage micrometer through the system allowed growth measurements to be taken directly from the screen. An on-screen running clock generated by the VCR gave very accurate time measurements. The proteins used in this study were lysozyme from hen egg white [4], canavahn from the Jack bean [5,6], leaf lectin II from Sophorajapomca [7], single strand D N A binding protein (SSB) from E~chertchla coh [8], and satellite tobacco mosaic virus (STMV) [9]. Lysozyme was dissolved in 0.1M sodium citrate at pH 4.9 to a concentration of 30 m g / m l . Thirty /~1 droplets were made from 15 ~1 of the protein solution and 15 /~1 of 8% w / v NaC1 also buffered at pH 4.9 with 0.1M citrate. Smaller droplets were made with proportionately less amounts of the two solutions The reservoir solutions were 8% w / v NaCI at pH 4 9 buffered with 0.1M citrate. Canavahn was dissolved in 1% w / v NaC1 to a concentration of 50 m g / m l upon addition of trace amounts of N H 4 O H to raise the pH to 8.5 to 9.5 Droplets of this canavahn solution could then be crystallized by vapor equilibration against reservoirs of 1% to 3% w / v NaC1 buffered to pH 6.8 with 0.075M sodium phosphate. Leaf lectin II from Sophora japontca was crystallized by equlhbratlng droplets initially containing 20 m g / m l protein buffered at pH 7.8 with 0.015M Tns-HCI also containing 8% w / w PEG 4000 against reservoirs of 14% w / w PEG 4000. SSB from E. coh was dissolved in 0.02M TrlsHC1 at pH 7.0 containing as well 0 30M NaC1. The protein concentration was 10 m g / m l . Droplets were crystalhzed by equilibration against reservoirs of 15% saturated sodium citrate. STMV, unbuffered at 20 m g / m l virus concentration and initially at 8% saturation with (NH 4) 2SO4, crystallized when equilibrated against 15% saturated ammonium sulfate. For sandwich drops, 15 /zl of protein solution was mixed with 15 ~1 of the precipitant solution and equilibrated against 0 5 ml of precipitant solution in the reservoir. Hanglng drop experiments were performed with 10 ~1 of protein solution being mixed with 10 /xl of

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• ;. Results a n d o b s e r v a t i o n s As seen from the plot of g r o w t h m fig 1, a ' y p l c a l crystal of l y s o z y m e grew rapidly and at a linear rate. A slight increase m g r o w t h rate was ,~bserved after 30 m m T h e rate of edge g r o w t h ',Lfter that t~me was calculated to be 840 A / s . This ~rystal a c c u m u l a t e d approxamately 24 layers of ~nolecules per second a n d achieved a length of 0.4 ~nm during 90 man of observation. A crystal of c a n a v a h n , not p a r t i c u l a r l y u n i q u e ~n terms of growth, also shown m fig. 1, grew at 1he r e m a r k a b l e rate of 1440 ,k,/s a c h i e w n g a length ~,f 0.5 m m m 76 min. This is a p p r o x i m a t e l y t w e n t y layers of t r l m e n c can a v a l in accruing to the surface 1,er second. In this case the g r o w t h rate is seen to , ary over the course of the e x p e r i m e n t T h e rate a acreased s o m e w h a t at 20, 40 a n d 60 n u n u t e s t,efore decreasing at 65 m i n u t e s w h e n the crystal p p r o a c h e d m a x i m u m size. In fig. 2, the g r o w t h rate of leaf l e c tm II ~s also seen to vary s o m e w h a t d u r i n g the course of the e x p e r i m e n t a c h i e w n g a m a x i m u m length of 0.11 ~ m in 21 h T h e g r o w t h rate d u r i n g the repreentatlve p e r i o d f r o m 3 to 6 h was m e a s u r e d to be 19 A / s , taking a b o u t 4 s for each layer of tetra-

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merlc lectin II ( m o l e c u l a r weight 130,000) to add to the surface of the crystal. A crystal of SSB p r o t e i n is shown in fig. 2 to grow at the rate of 58 A / s d u r i n g the time span f r o m 4 to 10 h b e f o r e l ev eh n g off at its m a x i m u m length of 0.34 ram. This rate c o r r e s p o n d s to app r o x i m a t e l y o n e layer of t e t r a m e n c SSB a d d i n g to the surface per second. In fig. 3, c a n a v a h n is sh o w n to grow u n d e r s~mllar c o n d i t i o n s at a m u c h slower rate than m the e x p e r i m e n t d e p i c t e d in fig. 1. In this case the rate of g r o w t h ~s e x t r e m e l y linear for the first 20 h

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S Koszelak et al / Protein crystal growth rates determined b) ttme lapse mtcrophotographv

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Fig 4 An example of a very slowly growing crystal is that of the lcosahedral virus STMV hawng a molecular weight of nearly two mdhon Nearly a full minute was necessar} for a crystal face to increase by one layer of virus particles before g r a d u a l l y decreasing as the crystal r e a c h e d its m a x i m u m length of 0.65 mm. T h e g r o w t h rate in the linear range is 73 A / s , or a p p r o x i m a t e l y one layer of trlmeric c a n a v a h n a d d e d to the surface p e r second. The difference in g r o w t h rates between this a n d the previous c a n a v a l l n e x p e r i m e n t m a y be a t t r i b u t e d to this crystal n u c l e a t i n g m u c h sooner after initiating the e x p e r i m e n t , thus the crystal e x p e r i e n c e d g r o w t h at a c o n s i d e r a b l y lower state of s u p e r s a t u r a t i o n t h a n that seen in fig 1. I n fig. 3, the g r o w t h of a crystal of S T M V is d e p i c t e d as well. A g a i n , the rate is n o t u n i f o r m over the time course of the e x p e r i m e n t . T h e rate in the range from 0.5 to 2.5 h was m e a s u r e d to b e 140 A / s , which is very r a p i d a n d c o r r e s p o n d s to slightly less than one w r a l p a r t i c l e a d d i n g to the surface per s e c o n d until a t e r m i n a l size was reached. In fig 4, a very slow rate of g r o w t h is seen for a second crystal of the virus. T h e rate of 3.1 A / s implies that over 50 s is r e q u i r e d for one layer of virus particles to be i n c o r p o r a t e d . T h e very different g r o w t h rates seen in this e x p e r i m e n t in c o n t r a s t to the one shown in fig. 4 m a y again be a t t r i b u t e d to the crystal nucleating sooner after initmtlng the e x p e r i m e n t , as well as a slightly lower initial virus c o n c e n t r a t i o n in the e x p e r i m e n t . A p e r i o d i c fluctuation in the rate of g r o w t h can also be seen in the graph. It shows a quite r e g u l a r p e r i o d of a b o u t 24 h. W e believe this m a y be due to the daily fluctuation In t e m p e r a t u r e of the r o o m where the e x p e r i m e n t was b e i n g c o n d u c t e d

In a d d i t i o n to q u a n t i t a t i v e m e a s u r e m e n t s of g r o w t h rates, several q u a l i t a t i v e o b s e r v a t i o n s have b e e n m a d e in e x a m i n i n g growing crystals b y h m e lapse p h o t o m i c r o s c o p y F r e q u e n t l y , crystals are o b s e r v e d to n u c l e a t e initially, i.e. at a lower level of s u p e r s a t u r a t i o n , on dust or a n y o t h e r sohd d e b r i s p r e s e n t in the sample. This effect is especially p r o n o u n c e d when using m i n e r a l crystals as h e t e r o g e n e o u s n u c l e a n t s [10,11] Such surfaces have also b e e n shown to direct crystal growth into a l t e r n a t e crystal unit cells. U s i n g time lapse w d e o m i c r o s c o p y , u n d e r c o n d i t i o n s f a v o n n g the formation of h e x a g o n a l crystals of c a n a v a l i n for example, a single crystal of the r h o m b o h e d r a l form of c a n a v a h n was o b s e r v e d to nucleate a n d grow on a piece of dust. The r e c o r d i n g showed that at a later time n u m e r o u s crystals of the h e x a g o n a l form then n u c l e a t e d in the s u r r o u n d i n g m o t h e r hquor. It was also o b s e r v e d that aggregates of crystals i n v a r i a b l y resulted from i n d i v i d u a l crystals coming together as the result of gravity or convective flow W e never o b s e r v e d crystals l n m a t l n g on the surfaces of others. T h e convective flow was directly o b s e r v e d in s o m e e x p e r i m e n t s when a small a m o u n t of p r o t e i n p r e c i p i t a t e was s i m u l t a n e o u s l y present. T h e flow of p r e c i p i t a t e suggested that the effects of c o n v e c t i o n can be quite p r o n o u n c e d . In one p a r t i c u l a r e x p e r i m e n t using c a n a v a h n , an u n u s u a l wave p a t t e r n o r i g i n a t i n g from a p o i n t source was seen to p r o p a g a t e across the surface of a large crystal. T h e origin was o b s e r v e d to be at the c o r n e r of a smaller crystal which h a d fallen on the surface of the larger a n d h a d b e c o m e i n c o r p o r a t e d as an inclusion. Such a p a t t e r n c o u l d be i n t e r p r e t e d as a series of m a c r o growth steps prod u c e d b y a screw d i s l o c a t i o n m e c h a m s m of crystal growth.

4. Discussion

T h e rate of g r o w t h of crystals typified by those shown here, varies s u b s t a n t i a l l y with each p r o t e i n a n d even for the s a m e p r o t e i n from one experim e n t to another. This is, of course, to b e expected since the rate of g r o w t h is a function of the degree of s u p e r s a t u r a t i o n [12,13], a n d that u n d o u b t e d l y was u n i q u e to each e x p e r i m e n t . Thus, a c o m p a r I -

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Protein ~rv~tal growth rates determmed by ttme htpse mlcrophotography

,,on of relative g r o w t h rates is i n a p p r o p r i a t e t h o u g h ,re are n o w a t t e m p t i n g to b e t t e r q u a n t i f y and ,neasure the degree of s u p e r s a t u r a t i o n so that such c o m p a r i s o n s are possible. In any case, it is clear that for some crystals ,uch as c a n a v a l l n an d lysozyme, the growth rates ,tre substantially tugher than anticipated, a n d core s p o n d in some cases to the a d d i t i o n of m a n y ayers of m o l ecu l es to a g r o w i n g crystal surface }~er second. Such rates of g r o w t h clearly suggest hat the rate of p r e s e n t a t i o n of molecules to crystal ~urfaces m ay have an i m p o r t a n t effect on the rate ,rod quality of crystal growth. It seems to s u p p o r t the c o n t e n t i o n that c o n v e c t i o n m a y play some role ~n those cases of rapidly g r o w in g crystals. Thus, l or such crystals, m l c r o g r a v l t y c o u l d exert a slgufacant influence. We frequently o b s e r v e d that small crystals sediJnentlng in the m o t h e r l i q u o r collided with, adl iered to, and, in m a n y cases, were i n c o r p o r a t e d )nto larger, faster g r o w i n g crystals. These pro([uced vistble i m p e r f e c t i o n s in the larger crystals. I n one case of c a n a v a l i n crystal growth, in fact, ,~'hat a p p e a r e d to be periodic succession of g r o w t h ~,teps could be seen p r o p a g a t i n g f r o m the p o i n t of J ncluslon of a small crystal into a large g r o w in g ~urface These o b s e r v a t i o n s f u r th e r suggest the l Legat~ve influence gravity has, due to sedamental Lon, on the crystalllzatton process. W e f o u n d it interesting that the p a t t e r n of row th characteristic of a particular p r o t e i n was J requently different for others. By e x p a n d i n g fur-

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ther the n u m b e r of p r o t e i n s e m p l o y e d in these e x p e r i m e n t s and by m o r e carefully c o n t r o l l i n g the e x p e r t m e n t a l variables, we h o p e to better define the n a t u r e of the p r o t e i n crystal g r o w t h p h e n o m e non. In so doing, we h o p e to discover n ew app r o a c h e s for its e n h a n c e m e n t and control.

References [1] D'Arcy Thompson, On Growth and Form, abridged 1961 edition (Cambridge Umverslty Press, Cambridge, 1917) [2] S Koszelak and A McPherson, J Crystal Growth 90 (1968) 340 [3] A McPherson, The Preparanon and Analysis of Protein Crystals (Wiley, New York. 1982) [4] C C F Blake. LN Johnson, GA Malr, A C T North. D C Phllhps and V R Sarma, Proc Ro,/ Soc (London) B167 (1967) 378 [5] A McPherson, J Blol Chem 255 (1980) 10472 [6] S C Smith, S Johnson, J Andrews and A McPherson, Plant Physlol 70 (1982) 1199 [7] A McPherson, C N Hanklns and L Shannon, J Blol Chem 262 (1987) 1791 [8] J D Ng and A McPherson, J Blomol Struct. Dynam 6 (1989) 1071 [9] S Koszelak, J.A Dodds and A McPherson, J Mol Blol 209 (1989) [10] A McPherson and P Schllchta, J Crystal Growth 90 (1988) 47 [11] A McPherson and P Schhchta, Science 239 (1988) 385 [12] R W Flddls, R A Longman and P D Calvert, Trans Faraday Soc 75 (1979) 2753 [13] M L Pusey and R Naumann J Cr?stal Growth 76 (1986) 593