The light-dependent modulation of photosynthetic electron transport

TIBS - February 1 9 8 3

52 sufficiently often that further discussion is warranted. Phosphatidic acid synthesis is catalysed by at least two membrane-bound enzymes: the well characterized sn-glycerol 3phosphate acyltransferase that catalyses the first acylation, and a second enzyme(s) that adds the second acyl chain 2~,22.There exists a large and conflicting literature concerning the specificity of the overall reaction towards saturated versus unsaturated chains and the effect of temperature on specificity21,22. It has been reported that the properties of the acyltransferase activity explain temperature-dependent control. However, these studies are flawed by the use of acyl-CoA substrates rather than acyl-ACP substrates. Recent results of Rock and Jackowski u indicate that acylACP, and not acyl-CoA, is the acyl donor for the incorporation of endogenously synthesized fatty acids into phospholipid. Moreover, the two acyl donors differ kinetically in vitroL The studies in vitro are also compromised by the use of unnatural acyl groups and quantitative effects too small to explain temperature-dependent contropO,21,22. There is, however, direct evidence for a second temperature-controlled reaction which functions only in the incorporation of exogenously added fatty acids from the medium into E. coli phospholipids. E. coli lives in an environment rich in fatty acids and utilizes these acids as a carbon source (by B-oxidation), and incorporates exogenous acids into its phospholipids, Sinensky 24 and Cronan s have shown that growth temperature alters the proportion of saturated and unsaturated fatty acids incorporated from an exogenously supplied mixture into phospholipid; the amount of saturated fatty acid incorporation increasing with increased temperature. Cronan ~ ruled out differential effects on transport, B-oxidation, or synthesis of endogenous acids as explanations for these results, These experiments therefore indicate a site of temperature control for the incorporation of exogenous acids. However, since the route of incorporation of these acids into phospholipid (following the activation with CoA accompanying transport) is unclear, the regulated step(s) is not known 2~'22. However, it is clear from the fabF mutant data that this mechanism is not involved in the temperature-dependent regulation of lipid composition in cells growing without exogenously supplied fatty acids. Conelusinns E. coli adjusts the fatty acid composition of its phospholipids in response to growth temperature. This regulatory mechanism functions to lower the temperature of the

order-disorder lipid phase transition and thus optimizes membrane function at the lower temperature. The regulatory mechanism is straightforward and is summarized in Fig. 2. Sensitivity to temperature is 'built in' to/3-ketoacyl-ACP synthase II. At low temperature this enzyme elongates the acyl-ACP ester of palmitoleic acid to cisvaccenic acid allowing formation of diunsaturated phospholipid species which lower the phase transition. At elevated temperatures, synthase II functions poorly and few diunsaturated phospholipids are formed. /3-Ketoacyl-ACP synthase II is required for temperature-dependent control because this enzyme is much more active than the other condensing enzyme, synthase I, in the elongation of palmitoleic acid to cisvaccenic acid. Teleologically, it would appear that E. coli acquired temperaturedependent control through the development of synthase II, a temperature-sensitive enzyme responsible for the synthesis of c/s-vaccenic acid. Acknowledgements This work was supported by NIH grant AI15650 and a postdoctoral fellowship (to DdM) from the Consejo Nacional de lnvestigaciones Cientificas y T6cnicas (CONICET) de la Reptiblica Argentina. DdM is a Career Investigator of CONICET.

teriol. Rev. 39, 232-256 4 Melchior, D. L. (1982) in Current Topics in Membranes and Transport (Razin, S. and Rottem, S., eds), Vol. 17, pp. 263-316, Academic Press 5 Marr, A. G. and lngraham, J. L. (1972) J. BacterioL 84, 1260-1267 6 Nishihara, M., Ishinaga, M., Kato, M. and Kito, M. (1976) Biochim. Biophys. Acta 376, 13-26 7 Baldassare, J. J., Rhinehart, K. B. and Silbert, D. F. (1976) Biochemistry 15, 2980-2994 8 Garwin, J. L. and Cronan, J. E., Jr (1980)J. Bacteriol. 141, 1457-1459 9 Cronan, J. E., Jr (1975) J. Biol. Chem. 250, 7074-7077 10 Okuyama, H., Yamada, K., Kameyana, Y., Ikezawa, H., Akamatsu, Y. and Nojima, S. (1977) Biochemistry 16, 2668-2673 11 Rock, C. O., Goelz, S. E. and Cronan, J. E., Jr (1981)J. BioL Chem. 256, 737-742 12 Polacco, M. L. and Cronan, J. E., Jr (1977) J. BioL Chem. 252, 5488-5490 13 Bell, R. M. (1974)J. Bacteriol. 117, 1065-1076 14 Cronan, J. E., Jr, Weisberg, L. W. and Allen, R. G. (1975)J. Biol. Chem. 250, 5835-5840 15 Gelmann, E. P. and Cronan, J. E., Jr (1972) J. BacterioL 112,381-387 16 D'Agnolo, G., Rosenfeld, I.S. and Vagelos, P. R. (1975)J. Biol. Chem. 250, 528%5294 17 Garwin, J. L., Klages, A. L. and Cronan, J. E., Jr (1980)./. Biol. Chem. 255, 3263-3265

18 de Mendoza,D., Garwin,J. L. and Cronan,J. E., Jr (1982)J. Bacteriol. 151, 1608-1611 19 Broekman, J. H. F. F. (1973)Thesis, University of Utrecht 20 Garwin, J. L., Klages, A. L. andCronan, J. E.,Jr (1980)J. Biol. Chem. 255, 11949-11956 21 Cronan, J. E., Jr (1978)Ann. Rev. Biochem. 47,

163-189 References 1 McElhaney,R. N. (1982) in Current Topics in 22 Rock, C. O, and Cronan, J. E., Jr (1982) in Current Topics in Membranes and Transport Membranes and Transport (Razin, S. and (Razin, S. and Rottem, S., eds), Vol. 17, pp. Rottem, S., eds), Vol..17,pp. 317-380, Academic Press 2 Quinn, P. J. (1981)Prog. Biophys. Molec. Biol. 38, 1-104 3 Cronan, J. E., Jr and Gelmann, E. P. (1975) Bac-

207-233, Academic Press 23 Rock, C. O. and Jackowski, S. (1982) J. Biol. Chem. 257, 1075%10765 24 Sinensky, M. (197t)J. Bacteriol. 106,449--455

The light-dependent modulation of photosynthetic electron transport N6stor Carrillo and Rub6n H. Vallejos Membrane-bound ferredoxin-NADP reductase undergoes light-driven conformational changes which favour its catalytic activity. Evidence is accumulating which suggests that these phenomena participate in the photoregulation o f photosynthetic electron transport and therefore in the regulation o f photosynthesis as a whole. It is well known that light, besides its function as the energy source for the photosynthetic fixation of CO2 in algae and higher plants, plays a major role in the regulation of photosynthesis. The H+-ATPase of chloroplasts and N~stor Carrillo and Ruben H. Vallejos are at the Centro de Estudios FotosintOticos y Bioquimicos, Suipacha 531, 2000 Rosario, Argentina.

,~ ElsevierBiomedicalPress 1983 0376- 5(k37/83/(~'~0- 0000/$0100

some of the key enzymes of the reductive Calvin cycle have been reported as targets of light-dependent modulation. Several mechanisms for the effect of light have been described; they involve conformational changes in membrane-bound proteins, an increase in the pH and Mg level in the chloroplast stroma, and redox changes 1. All these phenomena are mediated by

53

T I B S - February 1983

photosynthetic electron transport in addition to its basic functions, i.e. the production of reducing power in the form of NADPH, and the generation of an electrochemical potential across the membrane which is used for ATP synthesis. Both metabolites, i.e. NADPH and ATP, participate in the photosynthetic fixation of CO2. How is the electron transport activity affected by the numerous changes which occur in the chloroplast during dark-light transitions ? In this review, we summarize recent results which show that the photosynthetic electron transport chain is regulated by light at the level of ferredoxin-NADP reductase, via a combination of energy-driven and pH-mediated conformational movements of the membrane-bound enzyme, The photosynthetic electron transport chain Currently, the most accepted model for photosynthetic electron transport in organisms that evolve O2 is that in which two photosystems operate co-operatively in series, linked by a sequence of electron carriers which are asymmetrically located across the thylakoid membrane 2. Superimposed on this lineal, unidirectional redox chain, is a cyclic electron flow around photosystem 1 which involves some components of the lineal sequence, linked by a membrane bound cyt f-cyt bn complex. The emerging picture is summarized in Fig. 1. It is clear from Fig. 1 that Fd-NADP + reductase (FNR) holds a strategic position in the sequence of carriers, acting as a point of distribution of electrons which go either to the chloroplast stroma in the form of NADPH, or to the membrane again via the

NADP NADPkt. ~

"~,/2

O2+H÷

H20

l

kig. l. Schematic representation of the thylakoid tnembrane showitlg the prohahle distribution of the components of the photosynthetic electron transport chain.

cytochrome complex:L This membrane-bound flavoprotein was initially described 4 in 1956 as a NADPHspecific diaphorase, and was later studied thoroughly in its purified soluble form. Only recently has attention been focused on the membrane-bound form of the enzyme, i.e. the physiological form. The properties of ferredoxin-NADP reductase The purified soluble enzyme catalyses the reduction of NADP ' by Fd. In this system, reducing power is provided by atomic hydrogen generated in the medium by a kh.-hydrogenase system ~. The back reaction (i.e. the FNRcatalysed electron transfer from NADPH to Fd) takes place at high rates in the presence of a final acceptor like cytochrome c. The question of whether this back reaction occurs when the reductase is attached to the thylakoid membrane, was recently answered by Mills and co-workers'L They reported the existence of electron transport

from NADPH to plastoquinone, involving FNR. The reversible character of the FNRcatalysed reaction of thylakoids leads to an interesting observation: when the plant leaves are in the dark, it should be expected that some of the NADPH generated in the light (and not consumed during the reductive reactions of the Calvin cycle) should be oxidized again to N A D P as a consequence of this reversible electron transport. However, the NADPH/NADP ratio remains strikingly invariant in isolated chloroplasts during light/dark transitionsL This suggests that the back reaction does not occur significantly under physiological conditions. Moreover, experimental data indicate that electron transport in reverse can only be observed at significant rates under conditions of non-saturating light, but not in complete darkness". The data presented above suggest that there is, at some point in the electron tran~ port chain, a light-sensitive system which should remain inactive in the dark, thus avoiding the unnecessary waste of the NADPH generated during the light phase. The conformation of Fd-NADP ' reductase Different conformational states between soluble and membrane-bound reductases were first reported 8 in 1975. It was observed that the addition of the highly purified enzyme to FNR-depleted membranes resulted in a marked activation of its diaphorase activity. The allotopic properties of the reductase were later confirmed and extended by chemical modification experiments". Dansylation of the thylakoid membranes, for example, does not affect the diaphorase activity of the membranebound flavoprotein', while the soluble form is rapidly inactivated ~°. The pH profile for the diaphorase and FNR-mediated cytochrome c reductase activities of thylakoids is shown in Fig. 2.

54

TIBS - February 1983

Both activities increase rapidly as the pH is raised from 5.5 to 9.0, and have a pKa value of 7 (Ref. 11). Interestingly, these activity curves are similar to those reported '° ~z for the soluble enzyme, although the apparent pK, for the change in the soluble reductase is about 8. It has been suggested ~2 that the pH profile indicates a pH-dependent transition between two different conformational forms of the enzyme. An ionized SH group may be necessary for the high activity form, in both the soluble ''2 and membrane-bound reductases 1~ (Fig. 2). The chemical modification of enzymes is an important tool in the determination of the nature and biological function of amino acid residues in the active center. In the case of FNR, the participation of essential lysine and arginine was determined by the use of the specific reagents dansyl chloride and phenylglyoxal, respectively".10, ~. Treatment of thylakoid membranes with either reagent resulted in the inactivation of NADP + photoreduction in a time- and concentration-dependent manner. Surpri~ ingly, when modification was made in the light, higher rates of inhibition were obtained, suggesting that illumination increases the accessibility of the functional groups to the reagent. A catalyst of cyclic electron flow, e.g. pyocianine, was essential for such a phenomenon to occur, whereas uncouplers of photophosphorylation totally prevented itL These results indicate that light, via the formation of a transmembrane proton gradient, induces a conformational change in membrane-,

I

I

bound reductase which should affect polypeptide regions related to the active site. Does the observed conformational movement affect, in any way, the catalytic properties of the enzyme? The answer is yes. Protection afforded by N A D P against modification with several reagents was higher in the light than in the dark. A simple calculation of the kinetic data indicated that the dissociation constant of the membrane-bound enzyme for N A D P was increased by about one order of magnitude. by the illumination of the membrane in the presence of pyocianine" ~4. Even more illustrative are the results obtained by studying the variation in K,, for the physiological substrates N A D P and Ed, as a function of the energy state of the system. The K,,, values for both N A D P and Fd were drastically reduced when the light intensity was increased in well-coupled chloroplasts. Conversely, increasing the concentration of the uncoupler NH+CI produced the opposite effect ~" (Fig. 3).

The resultsobtainedwithisolated chloroplasts Light-dependent modulation of photosynthetic electron transport has been investigated independently in intact isolated spinach chloroplasts and in the green algae Bryopsis maxima, by Katoh and coworkers. Dark-adapted intact chloroplasts from algae and higher plants exhibit, on illumination with strong continuous light, sequen-

I

I

I

F

r

A

_2' c-" t.)

!~-0

10--

E > I=7"

5-

[~e

•

20

•

za, i--

I 0

I 6

I 7

I ~]

I 9

I 6

[ 7

I ~]

I g

0

pH tqg. 2. p H profile ]br diaphorase ( A ) a n d ~ytochrome c reductase (B) activities of.spinach thylakoids. Both activities were measured in a mixture o f buff~'rs containing 20 mM each o f 4-morpholinopropane sulfonic acid (MOPS), 4-morpholinoethane sulfimic acid ( M ES), and trk+ine-NaOtt at any tested pH. ( ontrol chloroplasts ( ~ ¢ ~ ) ; chloroplasts pretreated with I 0 m M N E M fi~r 2 hours prior to assay ( A - - &). This figure is m~',dified fi-om Ref. I 1.

tial and characteristic changes in the yield of chlorophyll a fluorescence, until a steady state is reached ]:'. The transient states observed during the first minutes of illumination, have been correlated with different manifestations of a temporary accumulation of reduced electronic carriers between photosystems I and ll. This accumulation is due to a blockage in the reducing side of photosystem 1, which is rapidly reverted by illumination. The site of dark-dependent inactivation was elegantly located by means of metabolites which accept electrons at different levels of the chain. NO.z, phosphoglycerate and oxalacetate were used, since they can permeate the double envelope of isolated chloroplasts. The site of dark-dependent inactivation was bypassed by NO.z which accepts electrons from Fd via nitrite reductase, but not in the presence of oxalacetate and phosphoglycerate which are reduced by NADPH. This suggests that the site of light-dependent modulation should be located at the level ofFNW ~'. Later studies revealed that the activation kinetics showed a half time of about 2 s, while the dark-dependent deactivation was much slower, and was completed in about 40 miri. Chloroplasts adapted to the dark have a considerable lag in NADP' photoreduction, and its diaphorase activity is markedly lower than in pre-illuminated chloroplasts ''+. What is the natureof the changes imposedby light on the membraneboundFNR? The nature of the light-induced change was analysed by flash kinetic spectroscopy. Purified enzyme was labelled with the fluorophore eosin-SCN before reconstitution on FNR-depleted thylakoids. In carefully controlled conditions it is possible to prepare a labelled flavoprotein which retains its catalytic activity and can be functionally reconstituted 17. When a suspension of eosin-labelled FNR-reconstituted thylakoid membranes is excited by a short pulse ofa Nd-YAG laser, a relatively stable triplet state is generated. The time dd~ay of such a triplet state is used to study the degree of exposure of the eosin binding sites to the bulk medium. Significant changes in the accessibility of the eosin binding sites were observed during dark-light transitions. This indicates that the membrane-bound protein undergoes a conformational change of the 'openingclosing' type TM.

How does the flavoproteindetectthe dark-lighttransitions? Two general hypotheses can be formulated to explain the mechanism of photo-

55

TIBS - February 1983.

5°L I

I

I

I

I

1

B

i ,° 10 0

t 0

40

I 80

I 120

I

I

2

4

LIGHt INTENSITY(ERG/CM2,S.IO tl)

NH4CI(mM)

big. 3. Effect oJlight intensity and Ntt4LI on the Km fi)r NA D P in N A D P photoreduction. In A, light intensities were as shown. In B, light intensity was saturating (I.5 ~ 10"erg.cm ~.s ') and N H ~( '1was added as stated ~. Similar results were obtained with ferredoxin 11

activation. The first mechanism is that the photoactivation of electron transport is related to light-induced changes in the energy state of the thylakoid membranes, or in the ionic composition of the stroma. The second, which is analogous to the mechanism of photoactivation of enzymes in the carbon-reducing cycle, assumes an involvement of light-generated and uncoupler-sensitive dithiols ~ in the activation of electron transport. The second mechanism can be ruled out, in view of the results obtained with isolated chloroplasts. For instance, suppression of photoactivation was achieved with the combined addition of 2,4-dinitrophenol and valinomycin, which have no effect on dithiol generation', Moreover, although FNR has a disulfide bond, its reduction does not alter the catalytic properties of the enzyme. Thus, we are now in a position to look for the first mechanism, and to distinguish whether the light activation is due to a primary structural change in the membrane-bound reductase driven by the

DARK

proton-motive force, or to a secondary alkalization of the stroma. The pH profile of Fig. 2 supports this second idea. The results obtained with intact chloroplasts also show a strict dependence of the photoactivation process on the stroma pH, in the expected manner'. However, according to the following observations, the light-dependent increase in affinity for NADP' and Fd cannot be explained in terms of changes in the proton concentration at the membrane-solution interphase: first, alkalization of the bulk medium results in an increase of the turnover of bound reductase without affecting the Km for N A D P and Fd t2. Second, similar light-dependent changes in affinity for both substrates were observed under a variety of pH values from 7.0 to 9.0 (Ref. 11). Therefore, the phenomena appear to be qualitatively different. One may speculate that both processes - the energydependent conformational change and the pH-driven transition - may contribute to give a highly active form of the enzyme under illumination, when more NADPH is

LIGHT

t
\

t:qO,us

d_J

needed for CO2 fixation. The key to understanding how the flavoprotein can detect the formation of a proton-motive force may necessarily lie in the association between protein and membrane. Binding of FNR to its site in the membrane results from a compromise between attractive Van der Waals forces and coulombic repulsion. When the surface charge is neutralized by a combination of cation binding and cation screening, the reductase becomes firmly bound to thylakoid membranes 2°. A direct interaction between the protein and the lipid bilayer is unlikely, since the thylakoid membrane is composed mainly of neutral lipids, thus precluding an electrostatic interaction. No evidence has yet been obtained concerning the possible existence of a protein acting as a membrane counterpart. Within this context, interesting observations were made on the rotational diffusion of membrane-bound FNR. The reconstituted enzyme rotates rapidly around an axis perpendicular to the membrane plane, with a time of relaxation of less than 1/.~s; this is drastically increased to about 40/.Ls when Fd is added. Fd is known to have a Specific interaction with FNR. The restriction in the free motion of the reductase cannot be ascribed to this interaction alone, since Fd is too small to cause such a dramatic effect, suggesting that FD may act as a bridge between FNR and another membrane component of higher molecular weight, presumably pbotosystem 1is (Fig. 4). Whatever its mechanism, the lightdependent change at the reducing terminal of the electron transport chain may have a regulatory function in photosynthesis. The photoactivation of the CO2-reducing cycle involves changes in activities of enzymes as well as changes in the ionic composition of the stroma, which are both dependent upon light-driven electron flow. Thus, electron transport must be activated first to bring the cycle into a functional state. Data reviewed here indicate that the light-dependent modulation of electron transport through FdNADP' reductase serves as an initial switch in the dark-light transition of photosynthesis.

E Acknowledgements This work was supported by grants from the Consejo Nacional de lnvestigaciones Cientificas y T6cnicas, Argentina. References

Fig. 4. Schematic representation o f the rotational motion o f eosin-labelled ferredoxin-NA D P reductase on the thylakoid membrane, in the absence or in the presence o f ferredoxin. This scheme is based on the results of Ref. 18.

I Buchanan, B.B., Wolosiuk, R . A . and Schfirmann, P. (1979)Trends Biochern. Sci. 4, 93-96 2 Golbeck, J. H., Lien, S. S. and San Pietro, A. (1977) in: Photosynthesis 1 (Trebst, A. and Avron, M., eds). pp. 94-116, Springer-Verlag

TIBS

56 3 Shahak, Y., Crowther, D. and Hind, G. (1981) Biochim. Biophys. Acta 636, 234-243 4 Avron, M, and Jagendorf. A. T. (1956) Arch. Biochem. Biophys. 65,475-490 5 Shin. M. and Arnon, D. 1. (1965)J. Biol. Chem. 240, 1405-1411 6 Mills, J. D., Crowther, D., Slovacek, R. E.. Hind, G. and McCarty, R. E. (19791 Biochim. Biophys. Acta 547. 127-138 7 Takahami, U., Shimitzu-Takahami. M. and Heber, U. (1981)Biochim. Biophys. Acta 637. 530-539 8 Schneemann, R. and Krogmann. D. W. (1975)

J. Biol. (_'hem. 250, 4965-497 I 9 Carrillo, N,, Lucero. H. A. and Vallejos, R. H. (1980) Plant Physiol. 65,495-498 10 Zanetti, G. (1976) Biochim. Biophys. Acta 445. 14-24 11 Carrillo, N., Lucero, H. A. and Vallejos, R. H. ( 1981 ) J. Biol. ('hem. 256, 1058-1 (159 12 Davis, D. J. and San Pietro. A. (1977) Arch Biochem. Biophys. 182,266-272 13 Zanetti, G., Gozzer. C.. Sacchi, G. and Curti, B. ( 1979 ) Biochim. Biophys. A eta 569, 127-134 14 Carrillo, N.. Arana, J, L. and Vallejos, R. H. ( 1981 ) J. Biol. ( hem. 256. 6823~5828

Crystallization of membrane proteins Hartmut Michel After a long period of fruitless attempts, three-dimensional crystals of membrane proteins are now available. 1"he best of the crystals are suited for high-resolution structure determination by X-ray crystallography. The use of small detergents and small amphiphilic molecules during crystallization pro red to he of great value. It is trivial to state that precise knowledge of the structure of biological macromolecules forms the basis for understanding their different functions and their molecular mechanism of action. Despite progress in different areas of structural research, cry~ tallography is still the only way to determine the spatial structure of proteins to atomic resolution. Structure determination using co-crystals of enzymes with inhibitors or substrates can then lead to the deduction of the mechanism of enzymecatalysed reactions. Crrystallography requires crystals, but there are two classes of protein, fibrous proteins and membrane proteins, which are inherently difficult to obtain in a suitable crystalline form. In the case of membrane proteins there was no three-dimensional crystal known until recently. Nevertheless, precise knowledge of their spatial structure is highly desirable, since they have many important functions, some of which are summarized below. (1) They catalyse the specific transport of metabolites and ions across membrane barriers. (2) They are involved in biological energy conversion; they convert the energy of sunlight into chemical and electrical energy, and they couple the flow of electrons to the synthesis of adenosinetripho~ phate, the universal high-energy intermediate of metabolism. (3) They act as signal receptors and transduce the signal across the membrane. The signal can be, for example, a neuroHartmut Michel is at the Max Planck Institute for Biochemist~. D-8033 Martinsried, F.R. (L

transmitter, a hormone, light or a chemotactic stimulus. As a special case of signal reception they are involved in cell-cell recognition. In none of these examples is the structure of the catalyst or its mechanism of action well understood. The best way to improve our knowledge concerning the structure and function of membrane proteins seems to be X-ray crystallography, since now, after a long period of fruitless trials, threedimensional crystals are available.

The problem Most biological membranes are made up of electrically insulating bilayers of lipids with the membrane proteins embedded in these bilayers. Those surfaces of the protein which are in contact with the alkane chains of the lipids must he highly hydrophobic, whereas those surfaces exposed to the aqueous phases on each side of the membrane are as hydrophilic as the surface of soluble proteins. Such an amphiphilic nature causes difficulties in the purification and crystallization of those typical membrane proteins which have large hydrophobic and hydrophilic surface areas.* In There are a few very hydrophohic membrane pro-reins with small hydrophilic domains like the lightharvesting chlorophyll proteins from photosynthetic bacteria or the N,N '.dicyclohexylcarbodiimidebinding protein from energy-transducing adenosinetriphosphatases. These are soluble in organic solvents and might be crystallized directly from these solutions. Crambin, a hydrophobic protein from Crarnbe abessynica, has been crystallized from ethanol. The structure of this probable membrane protein has already been determined at high resolution ~'.

- February 1983,

15 Satoh, Y. and Katoh, S. (198(I) Plant (ell Physiol. 21,907-916 16 Satoh. Y. (1981) Biochim. Biophys. Acta 638, 327-333 17 Wagner, R., Carrillo, N.. Junge, W. and Vallejos, R. H. (19811FEBS Lett. 131,335-340 18 Wagner, R., Carrillo, N., Junge, W. and Vallejos, R. H. (19821 Biochim. Biophys. Acta 680, 317-330 19 Yamagishi, A., Satoh, Y. and Katoh, S. (19811 Biochim. Biophys. Acta 637,264-271 2(I Carrillo, N. and Vallejos, R. H. (19821 Plant Physiol. 69, 21(~-213

contrast to globular proteins they are not soluble in aqueous buffer solutions, and for isolation and purification detergents must be added. Removal of the detergents leads to aggregation of the membrane proteins due to non-specific hydrophobic interactions. Detergents are lipid-like, amphiphilic molecules, which form micelles above a certain concentration (for properties of detergents see Refs 1-3). By the formation of micelles, direct contact between water and the alkane chains of the detergents is avoided. During solubilization of a membrane by an excess of detergent, the detergent micelles incorporate the lipids and the proteins. In some respect they replace the lipid bilayer. It is generally assumed that the alkane chains of the detergents are bound to the proteins' hydrophobic surface, whereas the polar parts of the detergents can interact with the proteins' polar surfaces. Detergents with charged polar head groups like dodecylsulfate or cetyltrimethylammonium frequently change the protein conformation and denature the protein. Using 'mild' detergents such as the polyoxyethylene detergents (e.g. Triton X100) or /3-D-octylglucopyranoside, many membrane proteins can be solubilized whilst retaining their native conformation. In any case the intactness of the protein should be proven by functional or spectroscopic tests. A tentative graphic model of membrane-protein solubilization with the help of detergents is shown in Fig. 1. The protein in the detergent micelle is then the starting material for purification and crystallization. The problem is to bring the protein into three-dimensional order taking into account the different properties of the hydrophobic and hydrophilic surface domains of the proteins. First reports on the crystallization of membrane proteins turned out to be irreproducible 4, and proofs or evidence that the material obtained was crystalline could not be presented s,'~. Only in the case of bacteriorhodopsin from the purple membrane of the halobacteria 7'8, and porin, an outer membrane protein from Escherichia coli 9,