Crystallization of membrane proteins: a minireview

Iournal of Crystal Growth 110 (1991) 89-95 North-Holland

89

,..• rystalllzat on of membrane proteins: a minireview °

°

R. M i c h a e l G a r a v a t o * a n d D a r n e l P i c o t 1~epartment of Biochemistry and Molecular Biology, The University of Chwago, 920 East 58th Street. Chicago. lllmols 60637, USA

Over ten years ago, the first reports appeared, demonstrating that membrane proteins could be crystalhzed [H Michel and D (~esterhelt, Proc, Natl Acad Scl USA 77 (1980) 1283, R M Caravlto and J P Rosenbusch, J. Cell Biol 86 (1980) 327] In the past d.~cade, other research groups have successfully prepared large single crystals of integral membrane protems for X-ray diffraction a aalys~s While no simple set of methods yet exasts, the general strategies for designing membrane protein crystalhzatlon experiments h 1re become clearer All X-ray quahty crystals of membrane proteins were grown from preparations of detergent-sohiblhzed protein. u ,lng standard crystallization methods for soluble proteins. In this article, we discuss the roles of the detergent, salt, and precipitant ~ the crystallization process and the adaptlon of pubhshed protocols to new membrane protein systems We focus on the p Ayethyleneglycol/NaC1/fl-octyl glucoside system as a general model and show that the presence of detergent applies several types o constraints to the crystalhzatlon conditions The general conclusion ~s that many integral membrane proteins could be crystallized 11 pure and monod~sperseprotein sohiuons in a statable detergent system can be prepared

1 Introduction The study of m e m b r a n e p r o t e i n structure has l( ,ng been f o r b i d d e n territory for X-ray crystallogr~Lphy simply because these proteins could n o t be c~ystallized. A m a j o r b r e a k t h r o u g h occurred in 1'180 with the crystallization of b a c t e r l o r h o d o p s i n [18] a n d E. cob p o r i n [8]. Since then, several l~ e m b r a n e proteins have been crystalhzed, a n d the structures of two of them, the p h o t o s y n t h e t i c reactt,)n centers from Rhodopseudomonas vtrldts a n d R~odobacter sphaerotdes, have been elucidated [1,5]. Recently, Picot a n d G a r a v i t o [23] have reported the first crystals of a m a m m a l i a n m e m h i a n e protein, p r o s t a g l a n d l n H synthase, that are araenable to high resolution X-ray analysis. As the n u m b e r of m e m b r a n e p r o t e i n crystals diffracting to high resolution is still small, it is too early to re Juce the crystalhzation process to a few general~sed steps. However, e x p e n m e n t a l observations h~ve revealed several r e q u i r e m e n t s for successful cr/stalllzatxon. I n this minirev~ew, the discussion is directed towards the status of crystalllzat~on m e t h o d s for m e m b r a n e proteins, as derived from * Fo whom correspondence should be addressed

the p u b h s h e d reports a n d reviews [1,3,9,11,12,19, 20,28,31], using the E cob p o r l n [12], the bacterial p h o t o s y n t h e t i c reaction centers [3,4] a n d prost a g l a n d i n H synthase [23] as the examples of the e x p e r i m e n t a l results. A n excellent review over the general m e t h o d s for purifying, handling, a n d crystallizing m e m b r a n e proteins has been recently p u b l i s h e d by K u e h l b r a n d t [15]. W h e n one wants to p l a n experiments on integral m e m b r a n e proteins, the gross structural characteristics of these proteins must be considered before e m b a r k i n g o n a crystalhzation project. Several features of m e m b r a n e p r o t e i n structure will affect p r o t e i n - d e t e r g e n t interactions and, therefore, i n f l u e n c e the crystallization process. Three m a j o r a s s u m p t i o n s have been proposed a n d have been m u c h discussed by the practitioners of m e m b r a n e p r o t e i n crystallization. While there is only c i r c u m s t a n t i a l evidence to s u p p o r t these suppositions, they are useful m e a n s for deciding on strategaes towards crystallizang a particular memb r a n e protein. Assumption 1: E x p e n m e n t a l observations suggest that as the extramembranous surfaces or domains of a m e m b r a n e p r o t e i n become larger a n d more d o m i n a n t , the less affected the crystalhza-

00-'2-0248/91/$03 50 '~ 1991 - Elsevier Science Publishers B V (North-Holland)

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R M GaraeltO, D Picot / Costalh:atton of membrane proteins a mmlrevle~t

tlon of that protein will be by the detergent In other words, more accessible protean surface provIdes more contact areas for crystalhzatlon. Proteans (or protein complexes like the photosynthetac reaction center [4] with large extramembranous domains might be "easy" to crystallize (i e. it would crystallaze more lake a soluble protean). On the other hand, a protein deeply embedded in the membrane (e g. E. colt porto) might require more subtle manipulation of the detergent environment Thus, the large saze of a receptor complex, such as the heterotetramenc human insulin receptor (Mr > 400,000 Da), would not necessarily be a disadvantage in crystalhzatlon experaments Assumptzon 2: Integral membrane proteins assocmte with the lipid bala?er m a number of different ways. Of Importance here is the nature of the transmembrane segments(s) It is proposed that an essentaal requarement for crystallazablhty is the existence of extramembranous protein domains on both sides of the bflayer to allow the three-dimensional contacts needed for crystal growth. However, it can be argued that membrane proteans with a single transmembrane segment (e g glycophorln) are less conformatlonally ragid than other integral membrane proteins (e.g the 7-hehx rhodopsin-class receptors) and are therefore less likely to be crystallazed. No real evidence exists to support (or disprove) thas assumption, though at is held by many researchers While the single membrane segment class of membrane proteins might be conformationally more flexible, a properly selected detergent environment might create a rigid enough detergent-protein complex that would allow crystalhzation. A~sumptton 3 As successful crystallization depends on the preparataon of pure, homogeneous protein, all factors that create chemical heterogeneity must be minimized or ehmmated Genetic ~,arlablhty must be dealt with by purifying protein from homozygous organasms or from geneucally distract expression systems. Posttranslatlonal modlftcatlons (e.g., glycosylahon or phosphorylatlon) that create a heterogeneous population of protein must also be dealt with While the chemical modifications themselves do not necessarily affect crystallazation, the heterogeneity they introduce will. Thus, a wise choice of expression systems and

expression condmons will dramatically improve the chances of successful crystallization

2. The general method The general theory and methods of crystalhzIng membrane proteans have been revaewed by Michel [21], Garavato et al [10] and most recently Kuehlbrandt [15]. Large crystals of integral membrane proteins have only been grown from monodlsperse, mlcellar solutions of detergent-solubihzed protein. Once a protein has been prepared in a suatable detergent system, the "classacal" methods for protein crystallization can then be used [16]. Both a m m o n i u m sulfate (AS) and polyethylene glycol (PEG) are effective crystallization agents in the presence of low concentrations (0.1-1.0% by volume or weight) of detergent. Microdaalysis and large-scale vapor diffusion have yielded the largest crystals [10,19], though macro-scale methods of sitting or hanging drop vapor diffusion afford quick and economical ways to test different crystallization condations. For the hanging drop method, at should be noted that the reduction m surface tension of the protein solution, due to the presence of detergent, limits the drop size to less than 5 /zL. The presence of detergents will modify other physical properties of the crystallizataon system. Protein-protein interactaons are the primary interactaons that also occur during crystalhzation. However, protein detergent and detergent-detergent interactions occur m membrane protein systems and must now be taken into account One can therefore expect that the crystallization process wall be influenced by the phase transltaons of detergents (e.g. the cloud point phenomenon and the subsequent separation of an aqueous solution into detergent-poor and detergent-rich phases). For example, detergent phase transitions, or even the approach towards a phase boundary, have been correlated with the crystallization of porln [10], or with the denaturation of the protein as is the case with bacteriorhodopsln [20]. Hence, the design of crystalhzatlon experiment~ is constrained by the phase behavior of the detergent as well as the protein. The phase transition bounda-

R M Garat, tto, D P t c o t / Cr~stalhzatton ofmernbraneprotems a mmlret,iew

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"'able 1 General condmons for membrane protein crystal growth Protein

Technique

Detergent

Addmve

Precipitant

Space group (resoluuon)

Ref

I'hotosynthetlc reaction center

Vapor diffusion

LDAO

Heptanemol

AS

P4~212 (2 5 A)

[19]

Vapor diffusion

LDAO fi-OG

PEG

P212121 (2 8 A)

[1,3]

Vapor diffusion

fi-OG

PCA

AS

P1 (4 A)

[18]

Vapor diffusion

LDAO

Heptanetrlol

PEG

P1, C222~ (10 ,~)

[28]

1 cob OmpF ponn

Microdialysls or vapor diffusion

fi-OG

C~E s

PEG

P42 (2 8 A)

[11]

t cob LamB

Vapor diffusion

fl-OG

PI, C2221

[12]

( Rsp t,trldls)

l'hotosynthetlc reaction center ( R sphaerotdes)

l ;acterlorhodopsm ( H halobmm) t' capsulatus hght harvesting

c :,mplex B8000-B850

PEG

(4 X) [a capsulatus Ponn

Vapor diffusion

CsE 4

PEG

R3 (3 2 ,~)

[35]

Prostaglandln H synthase (,)vine)

Vapor diffusion

fl-OG

PEG

I222 or I212121 (3 2 A)

[23]

( ytochrome c oxadase (t,ovme heart)

Ultrafiltratlon

Bnj 35

PEG

P62 or P64 (8 A)

[31]

Abbreviations AS ammonium sulfate, PEG polyethylene glycol, LDAO Dodecyl-dlmethylamine oxade, fl-OG fl-octyl glucosMe, C sE 5 octyl pentaoxylethylene; PCA, pipendme-2 carbonic acid

ries can be shifted by varying the lomc strength, tile protein concentration, and the concentration aad the kind of precipitating agents used for the c)3,stallizatlon. The addition of small amphlphihc c()mpounds at relatively high concentration [1 - 5 %] also has an influence on the crystallization process and the detergent phase transitions. While their u,,e has allowed the growth of large X-ray quahty ciystals of certain membrane proteins [4], the mode ol action of these "small amphaphiles" has not b(,'en adequately explained [10]. Summarized below are the factors considered to be the more crucial of the experimental "constraints" that are c~,rrelated to the appearance of macrocrystals and the eventual growth of large single crystals for X ray diffraction. (a) Puruy and homogeneity of the protem prep~ranon. A membrane protein must be first extracted from the m e m b r a n e and solubflized before crcstalhzatlon can be attempted. The solubflization step must keep the protein functional and

provade a basis for creating a monodlsperse, homogeneous protein-detergent complex that ~s sintable for b~ochermcal and biophysical characterization. Purification then becomes a most crucial step for crystalhzatlon: it must not only yield pure protein, but tt must also reduce all factors that cause molecular heterogeneity. For example, native hpMs are heterogeneous m composition and their removal ~s necessary m order to achaeve a homogeneous protein-detergent preparation. The presence of excess hpM, as well as detergent contamlnants, can be checked easily with thin layer chromatography (b) Detergent chowe. The choice of detergent(s) for purification and crystallization is quite important: the ones that have been used to grow crystals diffracting to high resolution are all nonionic and monodlsperse hke jS-octyl glucoslde (flOG), lauryl dlmethylamineoxade, decyl maltoslde (C10-M) or octyl pentaoxyethylene (see table 1 m ref. [10] or [15]). C o m m o n l y used detergents for

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R M Garavtto, D Picot / C~stalhzatlon of rnernbrane protems a mmtrevtew

solubihzation and for punfication (e.g., Tween-20 or Triton X-100) may allow some crystal formation, but have not yet provided any crystals diffracting to high resolution. If such detergents are used for protein preparation, they should be exchanged, at the end of the preparation, with another detergent more suitable for crystallization. In some cases, however, detergent exchange may be difficult and may require the use of other detergents for solubilizatlon (see next section). A question arises about the effect of lipids on crystalhzation. While heterogeneous native llpids have adverse effects on crystallization [9], one might ask if crystallization could occur in the presence of pure lipids. Most systems of phospholipids can not exist as a monodisperse micellar solution, but mostly as bilayer structures. We therefore do not expect crystal formation unless detergents are added to create a micellar solution However, some short chain phospholiplds form micelles (e.g. dioctanoyl-phosphorylchohne) and might be effective in allowing crystal growth. Elsele and Rosenbusch [7] have examined this hypothesis using E colt p o n n as a model system and observe good crystal growth. Thus, the ability of a pure surfactant, whether a detergent or lipid, to form a homogeneous micellar system may be the important physical criterion. Table 1 summanzes the published crystallization conditions for several membrane proteins. It should be noted that the conditions do not differ markedly from one another despite the wide variety of proteins examined. This implies that the experiences with other membrane proteins would provide good initial strategies, and initial conditions, for crystallization of novel Integral membrane proteins.

3. Detergent characteristics The key factor is crystalhzing membrane protelns is the judicious choice of detergent(s) for solubihzing the protein into a stable, monodisperse state. Three older reviews [13,14,27] cover, from a biochemical viewpoint, the action and behavior of detergents, while monographs by Tanford [26] and Rosen [25] as well as a review by

Wennerstrdm and Lindman [30] describe in detail the physical chemistry of detergents, rmcellar formatlon and solubflization. For crystallizing membrane proteins, the detergent must not only maintain the protein in a stable, solubilized state, but also must not inhibit the formation of protein-protein contacts that occur d u n n g crystal nucleation and growth. Thas aspect can be better appreciated by considering the molecular nature of a p r o t e i n detergent system. In an lsotropic, monodisperse solution of a m e m b r a n e protein in detergent, two distract rmcellar species exist, pure detergent mxcelles and mixed nucelles consisting of the protein partially surrounded by detergent. The latter species, which we wish to crystallize, is an amsotropic structure with two different surfaces (protein and detergent) exposed to the bulk solvent. The size, structure and behavior of the detergent layer around the protein depends on the character of the detergent(s) in it. Unfavorable detergent characteristics are major obstacles In membrane protein crystallization. The detergent factors winch can affect crystallization are" (1) Mtcelle size. A detergent layer of large physical s~ze around the protein can act as a barrier to close protein-protein contact. (2) Monomer flmdtty and mwelle deformablhty. The more fluid the detergent monomers are within the micellar region the more likely the micelle around the protein will be deformable. Hence, p r o t e i n protein interactions can distort the lmcellar surface, maximizing intermolecular contacts or exposing new contact s~tes (3) Mlcelle collotdal behawor. All detergents display concentration-dependent phase transitions. The simplest phase transition is just micellarizatlon. However, other phase changes can create nonisotropic solutions and detergent regions with distinct structural properties [22]. One common phenomenon which results in the eventual phase separation of the solution into two isotropic solutions is called the cloud point. Phasing out of the detergent is a frequent problem during crystalhzatlon experiments as this phase transition is easily induced by number of crystallization variables (e.g., detergent type, salt, temperature, and precipitant). The participation of protein-detergent mixed micelles in the aggre-

R M Garavtto, D Picot / C~stalhzatwn of membrane proteins a mmwevtew

gatlon process has not been well studied, but it is ,'lear that they partition into micelle aggregates ,rod, as shown by Bordler [2], they will partition ~nto the detergent phase when phase separation occurs. Weckstr~m [32] has studied the relation,~tup between phasing and the typical crystallizalion variables for Omp F porin crystallization. Small, pure and chemically well-defined deter~ents with moderate to tugh critical mlcellar cont'entrations (CMC) have turned out to be the best t'andldates for crystalhzation expenments. These detergents at concentrations above the CMC form Jsotroplc, monodisperse solutions of small nucelles (1.6-3.0 nm radius), thus resulting in a proteindetergent rmcelle with a total hydrodynarmc size ~Lot much larger than expected for the protein ~lone. Table 1 in Kuehlbrandt's review [15] lists a umber of detergents (and some of their physical characteristics), many of them frequently used m successful crystalhzatlon experiments. Choosing a c etergent remains a trial-and-error process and is complicated by the fact that not all membrane 1:roteins are stable in the best detergents for cryst dlization. A cursory glance at the list of detergents in I,~uehlbrandt [15] suggests that many classes of detergents have been explored. This is far from the t "uth as many detergents have been synthesized by c)emacal compames and abandoned because they had no commercial value in the consumer or industrial marketplace. Several classes of detergents have never been studied for their uesfulness in membrane biochemistry. Several interesting deterg mt classes have not been used for biochemical r~,'search: dimethylalkylphosphme oxides [33], alk gl betalnes [34], phosphobetaines [34], alkyl p hophorylcholines [29] and lysolecithin analogs [~9]. Weltzaen and collegues [29,35] have studied the last category of detergents and suggest detergents having a lysolecithin-like structure are excellent cadidates for membrane protein research.

4. A practical example: crystallization of prostaglandin H synthase Prostaglandin H synthase (PGS, EC 1.14.99.1) is an enzyme of the aractudonate cascade that

93

catalyzes the biosynthesis of prostaglandm H from arachidonlc acid [24], PGS is also the target of the so-called nonsteroldal anti-inflammatory drugs (NSAIDs). PGS is mostly localized in the endoplasmlc reticulum, but it has also been found m other membrane systems. Very little ~s known about the three-dimensional structure and the topology of the enzyme within the endoplasnuc membrane The recently published amino acid sequences [6,17] show the molecular size of PGS (600 amino acid residues, including signal sequence; 71000 Da, including glycosylation), but do not reveal potential intramembrane segments on inspection of the hydrophoblclty plots. However when PGS is solublhzed, the protein binds a considerable amount of detergent and lipid (up to 50% by weight). The glycosylation appears to be homogeneous between molecules and the putative glycosylation sites are located on the amino ternunil half of the protein. PGS is a reahstic model for eukaryotic membrane proteins and Picot and Garavito [23] have now shown that it is a good test case for the established crystallization methods. Picot and Garavlto [23] developed a purification scheme that was similar to earlier purification schemes except that the detergent Tween-20 was replaced by C~0-M. This was the critical change m the protocol: it allowed the preparation of a pure, monodisperse protein m a controlled detergent environment. Many of the steps in the purification, such as the D E A E 1on exchange chromatography, did not function solely to separate the PGS from other proteins, but also functioned to reduce the quantity of protein-bound lipids Reducing the hpid content in the PGS preparations to neghg~ble levels was a major factor in obtaining large crystals (ref. [23]; fig. 1). The detergent used for crystalhzation, fl-OG, is substituted for C~0-M by a molecular sieve step at the end of the preparation. The degree of delipadation and detergent exchange was monitored quahtatively by thin-layer chromatography (solvent system: chloroform/methan o l / a m m o m a 65 : 35 : 5 or water saturated 2butanone, visualization with iodine). Crystallization has been obtmned by vapor diffusion, using the hanging drop techmque: A 5 /~1 drop containing 15 m g / m l protein, 4% polyethyl-

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R M Garavlto, D Picot / C~'stalhzatmn of membrane protems a mtnzret,lew

ene glycol (MW 4000) and 0.6% fl-OG is equilibrated against a reservoir solution contaimng 8% to 16% polyethylene glycol. Small crystals may begin to appear after one day to one month after the start of the experiment depending on the condltions. The crystals (fig. lc) are rectangular or pseudohexagonal with a size about 0.5 × 0,3 × 0.1 mm. Their diffraction patterns extend beyond 3.5

resolution. They display orthorhomblc symmetry which suggests the space group I222 or I2~2121, and their umt cell dimensions are 97 × 208 × 229 A. If one dimer wtth detergent is assumed to have a molecular weight of about 240 kDa, a Vm of 2.4 A 3 / D a PGS per assymetric umt. Thas value is typical for crystals of protem-detergent complexes (R.M. Garavito, unpublished data). The apo-enzyme can also be crystallized under the same conditions as the holoenzyme (space group not determined). 13-OG is not unique m allowing the crystallization of PGS: some large crystals have also been grown with Ct0-M and octyl pentaoxyethylene.

5. Summary and conclusions Three-dimensional structure deterrmnatlon of membrane proteins had long been bandered by the apparent imposslbtlity of growing crystals suitable for X-ray diffractton. Recently, several membrane proteins have been crystalhzed and the three dtmenslonal structure of two of them solved to high resolution. It is clear that the methods developed for crystallmng membrane proteins can be applied to other systems, particularly energy transducing complexes or receptors. However, a substantial effort must be put into expressing and purifying the target protein to obtain a preparation that is homogeneous and monodlsperse by physical, chemical and genettc criteria There are

O

Fig 1 Effects of detergent and hpld contarmnanon on PGS crystal growth m fl-OG solutions C o n d m o n s of high lipid contarmnation causes the formation of needle-clusters of PGS crystals (a) m the presence of two hqmd phases (detergent-rich and detergent-poor phases) As the hpid content of the protein preparation decreases (b). the crystals become better formed and the larger show X-ray diffraction The detergent used for purifying this sample was Tween-20 and it poses a significant problem for the growth of better crystals as it can not be quantitatively removed from the sample When the hpld contaImnat~on becomes n u m m a l and the detergent environment homogeneous, large X-ray size crystals grow (c). Tins sample was purified m decyl maltoside and crystallized m fl-OG (see text) The largest crystals in (c) are about 0 4 m m long The magnification of panels (a) and (b) are 4 × and 2 × that of (c), respectively.

R M Garavtto, D P t c o t / C~'stalllzatlon ofmembraneprotems a mmzrevtew

ao a priori reasons that preclude the crystallization o f a n y p r o t e i n w i t h t r a n s m e m b r a n e

segments

a n d d e f i n e d e x t r a m e m b r a n o u s d o m a i n s if it c a n 3e o b t a i n e d as a p u r e , s t a b l e p r e p a r a t i o n i n a :mcellar d e t e r g e n t solution.

~,cknowledgements W e w o u l d h k e to a c k n o w l e d g e t h e i m p o r t a n t echnical support of N. Campobasso, M. Ludkow,ki a n d O. E k a b o i n o u r w o r k o n d e t e r g e n t c h a r ~ctenzation a n d P G S purification. W e also are ,~rateful to t h e N a t i o n a l I n s t t t u t e s o f H e a l t h a n d he Amertcan Cancer Society for their financial ,;upport of our research on E cob porin ( G M ~9550) a n d p r o s t a g l a n d i n s y n t h a s e ( B C - 5 8 1 ) . re,pectively

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[12] R M Garavlto, U Hmz and J-M Neuhaux, J Blol Chem 259 (1984) 4254 [13] A Helemus and K Slmons, Bloctum Blophys Acta 415 (1975) 29 [14] A Helemus, D R McCashn, E Fries and C Tanford, Methods Enzymol 56 (1979) 734 [15] W Kuehlbrandt, Quart Rev Blophys 21 (1988)4. [16] A McPherson, Preparation and Analysis of Protein Crystals (Wiley, New York, 1982) [17] J P Merhe, D Fagan, J Mudd and P Needleman, J Blol Chem 263 (1988) 3350 [18] H Michel and D Oesterhelt, Proc Natl Acad Scl USA 77 (1980) 1283 [19] H. Michel, J Mol B~ol 158 (1982) 567 [20] H Michel EMBO J 1 (1982) 1267 [21] H Michel, Trends Blochem Scl 8 (1983) 56 [22] D J Mitchell, C J T. Tlddy, L Wanng, T Bostock and M P McDonald, J Chem Soc Faraday Trans 79 (1983) 975 [231 D Picot and R M Caravlto, m Cytochroms P450 Biochemistry and Biophysics, Ed I Schulz (Taylor and Francis, London, 1989) p 29 [24] C R Pace-Asclak and W L Smith, Enzymes 16 (1983) 543 [25] M J Rosen, Surfactants and Interfaclal Phenomena (Wiley, New York, 1978) [26] C Tanford, The Hydrophoblc Effect (Wdey, New York, 1980) [27] C. Tanford and J A Reynolds, Blochlm Biophys. Acta 457 (1976)133 [28] W Welte, T Wacker, M Lels, W Kreutz, J Sluozawa, N Gad'on and C Drews, FEBS Letters 182 (1985) 260 [29] H U Weltzaen, C Rachter and E Ferber, J BIol Chem 254 (1979) 3652 [30] H Wennerstrom and B Llndman, Phys Rept 52 (1979) 1 [31] S Yoshtkawa, T Tera, Y Takahaslu, T Tsukthara and W S Caughey, Proc. Natl Acad Scl USA 85 (1988) 1354 [32] K Weckstrom, FEBS Letters 192 (1985) 220 [33] C C, Kresheck, Chem Phys Llplds 29 (1981) 69 [34] K W Hermann, J Colloid Interface Scl. 22 (1966) 352 [35] U Nestel, T Wacker, D WoltZlk, J Weckesser, W Kreutz and W Welte, FEBS Letters 242 (1989) 405