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Stabilizing membrane proteins
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  397 Membrane proteins can be extremely stable in a bilayerenvironment, but are often unstable and rapidly lose activityafter detergent solubilization. Poor stability can preclude thedetailed characterization of many membrane proteins. One wayto alleviate this problem is to find more stable mutants of amembrane protein of interest. This approach is made tractableby the finding that stability-enhancing mutations appear to berelatively common in membrane proteins. Addresses Department of Chemistry and Biochemistry, and UCLA-DOELaboratory of Structural Biology and Molecular Medicine, Boyer Hall,611 Charles E Young Drive East, Los Angeles, CA 90095-1570, USA;e-mail: bowie@mbi.ucla.edu Current Opinion in Structural Biology 2001, 11 :397–4020959-440X/01/$ —see front matter © 2001 Elsevier Science Ltd. All rights reserved. AbbreviationsBPTI bovine pancreatic trypsin inhibitor DGK diacylglycerol kinase PDB Protein Data Bank Introduction Although membrane proteins represent 20 to 30% of currentlysequenced genomes, only 0.2% of solved structures aremembrane proteins [1,2]. As a result, comparatively little isknown about how membrane proteins function and howtheir structure is defined by an amino acid sequence. Thegreat disparity between our understanding of soluble proteinsand membrane proteins has occurred largely because of themany practical problems of working with membrane proteins.One common problem is poor stability, which can lead torapid inactivation. Low stability renders many membraneproteins biochemically intractable and can precludehigh-resolution structure determination, for which a proteinmust be soluble at high concentrations and remain intactfor the days to weeks needed for crystals to form or forspectral data to be collected. Thus, it is essential that wedevelop methods to overcome this technical barrier if wehope to make more rapid progress in understandingmembrane protein structure and function. Why do membrane proteins die? To come to grips with the problem of the rapid inactivationof membrane proteins in detergent solution, it is important tounderstand the cause of inactivation, so that the appropriatedefects can be targeted for improvement. To this end, mycolleagues and I [3] have investigated the inactivationmechanism of the membrane-embedded enzyme diacyl-glycerol kinase (DGK). Our findings are summarized inthe pathway outlined in Figure1. First, we find that, atmodest temperatures, the inactive state is conformationallyaltered, rather than chemically modified. Somehow the proteinaccesses a low-energy conformation from which it cannotrecover. Second, the major pathway to the conformationallyaltered inactive state appears to involve transient dissociationof the folded, trimeric protein. The dissociated form of theprotein must be particularly susceptible to inactivation,either by exposing previously protected regions of the proteinor by increasing the flexibility of the protein chain, allowingaccess to stable, non-native conformations. Consistent withthe inactivation pathway we observed for DGK, dissociationof the cytochrome b  6  f  complex and Na + -K + -ATPase canalso lead to inactivation [4,5].The DGK inactivation pathway suggests that one way toslow membrane protein inactivation in detergent is tothermodynamically stabilize the folded, oligomeric form.This can be accomplished by adding compounds that bindto the native state, such as substrates or inhibitors [6]. Inaddition, it has become increasingly clear that lipid moleculesare often bound tightly to membrane proteins [7,8] andcan be useful additives for stabilizing membrane proteinsin detergent solution [9–11]. Finally, mutations that addstabilizing interactions in the folded form can reduce theconcentration of unfolded protein and thereby slowinactivation [3,12 • ,13,14]. New solubilizing agents Membrane proteins are designed to fold and function in alipid bilayer environment; in this environment, membraneproteins can be remarkably stable [15]. Thus, the idealmedium for working with membrane proteins is the lipidbilayer. Unfortunately, current technology for proteinpurification necessitates extraction fromthe membraneinto detergent micelles. To preserve the integrity of amembrane protein, the best we can hope to do is providean environment similar to the bilayer. The bilayer, however,is very complex, with large variations in lateral pressure,polarity and charge throughout [16,17 • ,18,19], and isessentially impossible to mimic very well. Consequently,detergent extraction is likely to be, at best, mildly destabi-lizing. As discussed above, in the case of DGK, it is thisdestabilization that is the primary cause of inactivation [3].The trick is to find a detergent each protein can tolerateand efforts continue to find new detergents.Yu et al. [20 • ] have recently developed a particularlyinteresting detergent series called tripod amphiphilesbecause of the three hydrophobic tails emanating from thepolar head group. The tripod amphiphile shown inFigure2 was capable of maintaining bacteriorhodopsin andbovine rhodopsin in a soluble, folded conformation formany weeks. Because the apolar domain of thisamphiphile is relatively short, it may produce a smalleramphiphile doughnut around the transmembrane region of the protein compared with the more commonly used Stabilizing membrane proteins James U Bowie  single-chain detergents. This smaller micelle could facilitatetype II crystal formation [21,22].Amphipols, introduced by Tribet et al. [23], are a completelynovel concept for membrane protein solubilization. Amphipolsare mixed copolymers that possess a hydrophilic backboneand hydrophobic sidechains (see Figure3) [23]. It isthought that the amphipol can wrap around the hydrophobicportions of a membrane protein, exposing the amphipol’shydrophilic backbone to the aqueous environment. Intheory, amphipols could have significant advantages overdetergent. In detergent-solubilized membrane proteinsolutions, much of the detergent is present as emptymicelles or free monomers. Free detergent micelles andmonomers complicate work with membrane proteinsbecause they can drive dissociation of bound hydrophobicmolecules or dissociation of subunits into empty micelles;lead to phase separation problems during crystallization; andincrease sample viscosity in NMR experiments [23]. Theamphipol–protein complex can be sufficiently stable thatfree amphipol is essentially nonexistent. The amphipol actsmore like an additional, nonprotein subunit. Freedom fromdetergent solutions could even enable the use of membraneprotein–amphipol complexes as injectable drugs — some-thing that would be impossible with detergent-solubilizedprotein. Current amphipols can maintain the solubility of some membrane proteins in the absence of detergent, butare still not a quantum leap over detergents in maintainingnative protein in aqueous solution [23,24]. Hopefully,further development of this interesting concept will yieldstill more effective compounds. Stability-enhancing mutations are not rare Although it is not possible to consider the rationalengineering of a membrane protein for enhanced stabilityin the absence of its three-dimensional structure, it appearsthat stability-enhancing mutations are not rare, such thatdesign may not be necessary. For example, Deber andco-workers [25,26] found two valine to alanine substitutionsin a stretch of 19 residues in the transmembrane segmentof the M13 coat protein that improved thermostability.Similarly, in a set of 20 cysteine substitutions introducedinto a transmembrane segment of DGK, two were foundthat significantly increased the half-life in detergent solution[13]. Perozo and co-workers [14,27] measured the stabilityof KcsA K + channel mutants by monitoring the rate of irreversible subunit dissociation at different temperatures.In a large set of 66 cysteine substitutions, eight enhancedstability more than 1 kcal/mol per subunit. Finally, one outof nine mutations in helices B and D of bacteriorhodopsinincreased stability (T Isenbarger, M Krebs, personal com-munication). Thus, it appears that roughly 10% of randommutants will improve the stability of a membrane protein— a remarkably high number. By comparison, in a set of 45alanine substitutions introduced into the soluble proteinBPTI, none increased stability, and of 51 alanine substitutionsintroduced into the soluble Arc repressor protein, only oneimproved stability [28,29]. Lack of a high level of stability optimization The fact that a high frequency of stabilizing mutations canbe found indicates that many stabilizing interactions havegone untapped in membrane proteins. This indicates thatmembrane proteins are not highly optimized for stability.Consequently, it is relatively easy to stumble onto theseunused stabilizing interactions by accident. There are severalpossible reasons for this lack of stability optimization.First, if flexibility is required for a protein’s function [30],high stability could reduce activity. The need for proteinmobility may explain why many thermophilic membraneproteins are only functional at high temperature. The factthat stable mutants of DGK and bacteriorhodopsin possesshigh levels of activity argues against this possibility, butsmall activity differences may be relevant in the wild.Second, protein turnover in the cell may require moderationof stability. For soluble proteins, thermodynamic stability can 398 Membranes Figure 1 Predominant inactivation pathway of DGK. Folded trimerMonomer Current Opinion in Structural Biology Stable alteredconformation/aggregate Figure 2 Chemical structure of a tripod amphiphile. NH Current Opinion in Structural Biology +NOO    be closely correlated with turnover rate in the cell, becauseunfolded proteins are more susceptible to proteolysis [31]. If a protein is too stable, it may be difficult to degrade, resultingin selective pressure to moderate stability. To my knowledge,a correlation between degradation rate and thermodynamicstability has not been examined for membrane proteins, so itis difficult to assess the likelihood of this hypothesis.Third, stability increases are usually observed in detergentsolution, not in the natural environment. It is possible thatmembrane proteins are indeed highly optimized for themembrane environment, but not for the micelle environ-ment. Isenbarger and Krebs (T Isenbarger, M Krebs, personalcommunication), however, have shown that the stabilizingmutation of bacteriorhodopsin is effective in the naturalpurple membrane. Moreover, we find that seven out of eight mutants of DGK that the slow inactivation ratein detergent also slow the inactivation rate when recon-stituted into membrane vesicles (D Yang, JU Bowie,unpublished data).Finally, because of the many conformational restrictionsimposed on a protein that is inserted into a lipid bilayer, ahigh degree of optimization may not be requiredto achievea level of stability that is sufficient for cell viability. When amembrane protein inserts into the bilayer, the transmembranehelical structure and topology is largely fixed, and eachtransmembrane helix has restricted motion about thebilayer normal [32,33]. As a result, the number of possibleconformations is relatively modest, so it may not be difficultto construct a sequence that favors a single conformation.As selective pressure will exist only to achieve a level of stability needed for cell viability, many potential stabilizinginteractions may go unused in natural membrane proteinsequences, because they are not needed. By comparison,soluble proteins must favor one conformation over a hugenumber of possible unfolded conformations. Consequently,a higher level of optimization may be required for solubleproteins than for membrane proteins. This would explainthe relatively low frequency of stabilizing mutations foundin the soluble BPTI and Arc repressor proteins. Building hyperstable membrane proteins Because stabilizing mutations are not hard to find, mycolleagues and I [12 • ] tested the possibility of doing arandom screen for stabilizing substitutions of DGK. TheDGK gene was mutagenized and individual clones weregrown in the wells of a microtiter plate. The cells werethen pelleted and the membranes solubilized in detergent.One aliquot was assayed directly for DGK activity and asecond aliquot was heated to inactivate the protein. Cloneswith higher activity remaining after the heat treatmentthan the wild-type protein were selected. In this manner,we identified a collection of 12 different single mutants of DGK with improved longevity in detergent solution.We then tested whether the mutations could be combinedto yield even more stable variants. Each additional mutationimproved stability, indicating that the stabilizing effectsare at least partially additive. We ultimately constructed aquadruple mutant that increased the half-life in n-octyl-glucoside from 6 min at 55 ° C for the wild-type protein to35 min at 80 ° C for the quadruple mutant. The quadruplemutant retains a high specific activity that is about two-thirdsthat of the wild-type enzyme. By adding more stabilizingmutations, it is likely that stability could be improved evenfurther. Thus, it is possible to convert a marginally stableprotein into an extremely robust one. Improved stabilitycan certainly make membrane proteins easier to work within the laboratory. Moreover, because such hyperstablemembrane proteins could withstand harsh conditions andhave a long shelf life, they may ultimately prove useful inindustrial processes or even as injectable drugs. Auspicious sites for mutations In our library of stability-enhancing mutations of DGK,we found many positions where multiple amino acidsubstitutions improved stability [12 • ]. This suggests thatthere are hot spots in the sequence for stabilizing mutations.These hot spots may occur where the wild-type residue isdestabilizing. If it were possible to identify likely hot spots Stabilizing membrane proteins Bowie 399 Figure 3 Amphipols. (a) Chemical structure of the amphipol polymer. The polymercan be made with different ratios (X, Y and Z) of monomer units. (b) A hypothetical depiction of a protein–amphipol complex. Thehydrophobic sidechains of the polymer (yellow)are sequestered againstthe hydrophobic region of the membrane protein, leaving the hydrophilicbackbone (red) in contact with water. (a)(b) CH 2 CH 2 CH 2 CHCHCHCOCOCO 2 NHNH Current Opinion in Structural Biology YZX  inthe sequence, these sites could be targeted for mutagenesis,improving our chances of obtaining stable mutants.Examination of the limited available data suggests at leasttwo possible trends.First, sites where stability-enhancing mutations are foundappear to be more prevalent in the interfacial region of thebilayer. Figure4 shows the positions of known stability-enhancing mutations in membrane proteins. Althoughstabilizing mutations can occur anywhere, the majority arenear the edge of the bilayer. It is possible that pinningdown the ends of transmembrane helices improves tertiarystructure stability because, with stable, rigid helices,locking the ends may define the structure better thanlocking the middles.Second, stabilizing mutations are apparently more likelyto occur at the β -branched residues isoleucine and valine.Of the 19 sites where stabilizing mutations have beenidentified in the proteins discussed above, for nine sitesthe wild-type residue is isoleucine or valine (47%). Bycomparison, the frequency of isoleucine/valine residues atall positions in these proteins is only 20%. Thus, it appearsmore likely that a mutation at an isoleucine or valineresidue will result in higher stability compared with otherresidues. Why might this be the case? One possibility isthe low helix propensity of isoleucine and valine residues,which could possibly destabilize the helices. It is thoughtthat transmembrane helix-coil transitions play little role inmembrane protein tertiary structure stability, however,because helices are so stable in the hydrophobic bilayer ordetergent micelle, where unfolded backbone hydrogenbonds can not be satisfied by hydrogen bonds to water[15,16,32,34]. On the other hand, most of these mutationsoccur in the interfacial region. Because water can penetratethis region, helix-coil transitions may play some role inoverall stability, although even in the interface an unsatisfiedbackbone hydrogen bond is costly [16,19]. Another possibleexplanation is that the β -branch leads to limitations of thesidechain conformations in a helical structure [35]. As aresult, the sidechain may not be as capable of adjusting tooptimize packing. For example, in their modeling of the M13 coat protein, Wang and Deber [25] found that thestabilizing V31A mutation allowed closer packing of thehelices than was possible in the wild-type sequence.Certainly, more data are needed to establish the generalityof these trends and perhaps to identify more criteria forsite selection. Designing stability enhancement Membrane proteins have unique properties that couldmake them practically useful for chemical separations,biosensing or even as pharmaceuticals [36]. For theseindustrial applications, however, it will be important todevelop robust membrane proteins that can withstandharsh conditions or possess a long shelf life. To this end,rational design strategies may become important.Efficient algorithms for the optimization of core packinghave been developed for soluble protein design [37].These design strategies could be directly applicable tomembrane proteins, for which packing may play adominant role in structural stabilization. Recent workdemonstrating the strength of hydrogen bonding in themembrane argues that an effective strategy for engineeringstability would be to design specific hydrogen bonds into amembrane protein structure [38 • –40 • ]. Finally, theintroduction of disulfide bonds may be a simple andeffective strategy for improving stability. For example,Oprian’s group [41] has been able to engineer disulfides intorhodopsin based on only a crude structural model. Conclusions Membrane proteins are often thought of as delicateweaklings, unable to withstand the rigors of life outsidethe safety of the bilayer. Consequently, experimentalistsgenerally avoid them if possible and practical uses of mem-brane proteins are rarely even considered. Nevertheless,low stability appears to be more of a technical problemthan a problem inherent to their make-up or the unique 400 Membranes Figure 4 The positions of stabilizing mutations in thebilayer. The structures of (a) bacteriorhodopsin(PDB code 1c3w) and (b) the KcsA potassiumchannel (PDB code 1bl8) are shown as blueribbons. The retinal chromophore ofbacteriorhodopsin and the bound ions of KcsAare shown in yellow. The two lines represent theapproximate span of the membrane. The mosthydrophobic 30 slicesperpendicular to themembrane normal of bacteriorhodopsin andKcsA are positioned between the lines. Topologydiagrams of (c) DGK and (d) the M13 coatprotein are also shown. The positions ofstabilizing mutations are shown in red. For KcsA,only mutants that enhanced stability more than 1kcal/mol per subunit are included. For DGK, onlythe positions of mutants that improved the half-life more than threefold are shown. (a)(b)(c)(d) 30 Å Current Opinion in Structural Biology  environment in which they reside. As we developimproved methods for stabilizing membrane proteins,these attitudes may change, leading to an improved under-standing of membrane protein structure and function, andexpanded practical uses of their unique properties. Acknowledgements The author would like to thank Aaron Chamberlain, Salem Faham andSarah Yohannan for comments on the manuscript, and the NationalInstitutes of Health for support (grants GM59164 and GM63919). References and recommended reading Papers of particular interest, published within the annual period of review,have been highlighted as: • of special interest •• of outstanding interest1.Wallin E, von Heijne G: Genome-wide analysis of integralmembrane proteins from eubacterial, archaean, and eukaryoticorganisms. Protein Sci 1998, 7 :1029-1038.2.Boyd D, Schierle C, Beckwith J: How many membrane proteins arethere? Protein Sci 1998, 7 :201-205.3.Zhou Y, Lau FW, Nauli S, Yang D, Bowie JU: Inactivationmechanism of the membrane protein diacylglycerol kinase indetergent solution. Protein Sci 2001, 10 :378-383.4.Breyton C, Tribet C, Olive J, Dubacq JP, Popot JL: Dimer tomonomer conversion of the cytochrome b6f complex. Causesand consequences. J Biol Chem 1997, 272 :21892-21900.5.Esmann M: Solubilized (Na ++ K + )-ATPase from shark rectal glandand ox kidney—an inactivation study. Biochim Biophys Acta 1986, 857 :38-47.6.Zhou Y, Wen J, Bowie J: A passive transmembrane domain. Nat Struct Biol 1997, 4 :986-990.7.Tsukihara T, Aoyama H, Yamashita E, Tomizaki T, Yamaguchi H,Shinzawa-Itoh K, Nakashima R, Yaono R, Yoshikawa S: The wholestructure of the 13-subunit oxidized cytochrome c oxidase a 2.8 Å. Science 1996, 272 :1136-1144.8.Luecke H, Schobert B, Richter H, Cartailler J, Lanyi J: Structure ofbacteriorhodopsin at 1.55Å resolution. J Mol Biol 1999, 291 :899-911.9.Lund S, Orlowski S, de Foresta B, Champeil P, le Maire M, Moller JV: Detergent structure and associated lipid as determinants in thestabilization of solubilized Ca 2+ -ATPase from sarcoplasmicreticulum. J Biol Chem 1989, 264 :4907-4915.10.Levi V, Rossi JP, Echarte MM, Castello PR, Gonzalez Flecha FL: Thermal stability of the plasma membrane calcium pump.Quantitative analysis of its dependence on lipid-proteininteractions. J Membr Biol 2000, 173 :215-225.11.Callaghan R, Berridge G, Ferry DR, Higgins CF: The functionalpurification of P-glycoprotein is dependent on maintenance of alipid-protein interface. Biochim Biophys Acta 1997, 1328 :109-124.12.Zhou Y, Bowie J: Building a thermostable membrane protein. J Biol • Chem 2000, 275 : 6975-6979 .This paper describes the construction of a robust membrane protein fromone that was marginally stable.13.Lau F, Nauli S, Zhou Y, Bowie J: Changing single side-chains cangreatly enhance the resistance of a membrane protein toirreversible inactivation. J Mol Biol 1999, 290 :559-564.14.Perozo E, Cortes DM, Cuello LG: Three-dimensional architectureand gating mechanism of a K + channel studied by EPRspectroscopy. Nat Struct Biol 1998, 5 :459-469.15.Haltia T, Freire E: Forces and factors that contribute to the structuralstability of membrane proteins. Biochim Biophys Acta 1995, 1228 :1-27.16.White SH, Wimley WC: Membrane protein folding and stability:physical principles. Annu Rev Biophys Biomol Struct 1999, 28 :319-365.17.Curran AR, Templer RH, Booth PJ: Modulation of folding and • assembly of the membrane protein bacteriorhodopsin by inter-molecular forces within the lipid bilayer. 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Proc Natl Acad Sci USA 1993, 90 :11648-11652.27.Cortes DM, Perozo E: Structural dynamics of the Streptomyces lividans  K + channel (SKC1): oligomeric stoichiometry and stability. Biochemistry 1997, 36 :10343-10352.28.Milla ME, Brown BM, Sauer RT: Protein stability effects of acomplete set of alanine substitutions in Arc repressor. Nat Struct Biol 1994, 1 :518-523.29.Yu MH, Weissman JS, Kim PS: Contribution of individual side-chains to the stability of BPTI examined by alanine-scanningmutagenesis. J Mol Biol 1995, 249 :388-397.30.Joseph D, Petsko GA, Karplus M: Anatomy of a conformationalchange: hinged ‘lid’ motion of the triosephosphate isomeraseloop. Science 1990, 249 :1425-1428.31.Parsell DA, Sauer RT: The structural stability of a protein is animportant determinant of its proteolytic susceptibility in Escherichia coli  . J Biol Chem 1989, 264 :7590-7595.32.Popot J, Engelman D: Membrane protein folding and oligomerization:the two-stage model. Biochemistry 1990, 29 :4031-4037.33.Bowie J: Helix packing in membrane proteins. J Mol Biol 1997, 272 :780-789.34.Deber CM, Li S-C: Peptides in membranes: helicity andhydrophobicity. Biopolymers 1995, 37 :295-318.35.Blaber M, Zhang XJ, Lindstrom JD, Pepiot SD, Baase WA,MatthewsBW: Determination of alpha-helix propensity within thecontext of a folded protein. Sites 44 and 131 in bacteriophage T4lysozyme. J Mol Biol 1994, 235 :600-624.36.Braha O, Gu LQ, Zhou L, Lu X, Cheley S, Bayley H: Simultaneousstochastic sensing of divalent metal ions. Nat Biotechnol 2000, 18 :1005-1007.37.Desjarlais JR, Clarke ND: Computer search algorithms in proteinmodification and design. Curr Opin Struct Biol 1998, 8 :471-475.38.Zhou FX, Cocco MJ, Russ WP, Brunger AT, Engelman DM: • Interhelical hydrogen bonding drives strong interactions inmembrane proteins. Nat Struct Biol 2000, 7 :154-160.A clear demonstration of the strength of hydrogen bonds in the bilayer. A sin-gle asparagine residue can drive the association of a transmembrane helix.The authors suggest that the presence ofpolar residues in membrane pro-teins may be dangerous because they could lead to strong, inappropriateinteractions with other proteins. Stabilizing membrane proteins Bowie 401
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