How Membranes Shape Protein Structure

membrane protein transmembrane interface hydrocarbon core dioleoylphosphatidylcholine palmitoyloleoylphosphatidylcholine Constitutive α-helical membrane proteins (MPs)1 are assembled in membranes by means of a translocation/insertion process that involves the translocon complex (1Johnson A.E. van Waes M.A. Annu. Rev. Cell Dev. Biol. 1999; 15: 799-842Crossref PubMed Scopus (499) Google Scholar). After release into the membrane's bilayer fabric, a MP resides stably in a thermodynamic free energy minimum (evidence reviewed in Refs. 2Lemmon M.A. Engelman D.M. Q. Rev. Biophys. 1994; 27: 157-218Crossref PubMed Scopus (177) Google Scholar and 3White S.H. Wimley W.C. Annu. Rev. Biophys. Biomol. Struct. 1999; 28: 319-365Crossref PubMed Scopus (1472) Google Scholar). This means that the prediction of MP structure from the amino acid sequence is fundamentally a problem of physical chemistry, albeit a complex one. Physical influences that shape MP structure include interactions of the polypeptide chains with water, each other, the bilayer hydrocarbon core, the bilayer interfaces, and cofactors (Fig.1). Two recent reviews (3White S.H. Wimley W.C. Annu. Rev. Biophys. Biomol. Struct. 1999; 28: 319-365Crossref PubMed Scopus (1472) Google Scholar, 4Popot J.-L. Engelman D.M. Annu. Rev. Biochem. 2000; 69: 881-922Crossref PubMed Scopus (535) Google Scholar) provide extensive discussions of the evolution, structure, and thermodynamic stability of MPs. Here we provide a distilled (and updated) overview that addresses four broad questions. What is the nature of the bilayer matrix that encloses MPs? How can the thermodynamic principles of MP stability be discovered? How does the bilayer matrix induce structure? How can the structure of MPs be predicted? We focus primarily on α-helical proteins, but the thermodynamic principles we present also apply to β-barrel MPs, which Lukas Tamm discusses elsewhere in this series. Two influences will emerge as paramount in shaping MP structure. First, as implied in Fig. 1, the bilayer fabric of the membrane has two chemically distinct regions: hydrocarbon core (HC) and interfaces (IFs). Interfacial structure and chemistry must be important, because the specificity of protein signaling and targeting by membrane-binding domains could not otherwise exist (5Hurley J.H. Misra S. Annu. Rev. Biophys. Biomol. Struct. 2000; 29: 49-79Crossref PubMed Scopus (225) Google Scholar). Second, the high energetic cost of dehydrating the peptide bond, as when transferring it to a non-polar phase, causes it to dominate in the formation of structure (6Liu Y. Bolen D.W. Biochemistry. 1995; 34: 12884-12891Crossref PubMed Scopus (423) Google Scholar). The only permissible transmembrane structural motifs of MPs are α-helices and β-barrels, because internal H-bonding ameliorates this cost. Because membranes must be in a fluid state for normal cell function, only the structure of fluid (Lα-phase) bilayers is relevant to understanding how membranes mold proteins. However, atomic resolution images of fluid membranes are precluded because of their high thermal disorder. Nevertheless, useful structural information can be obtained from multilamellar bilayers (liquid crystals) dispersed in water or deposited on surfaces. Their one-dimensional crystallinity allows the distribution of matter along the bilayer normal to be determined by combined x-ray and neutron diffraction measurements (liquid crystallography; reviewed in Refs. 7White S.H. Wiener M.C. Disalvo E.A. Simon S.A. Permeability and Stability of Lipid Bilayers. CRC Press, Boca Raton, FL1995: 1-19Google Scholar and 8White S.H. Wiener M.C. Merz K.M. Roux B. Membrane Structure and Dynamics. Birkhäuser, Boston1996: 127-144Google Scholar)). The resulting "structure" consists of a collection of time-averaged probability distribution functions of water and lipid component groups (carbonyls, phosphates, etc.), representing projections of three-dimensional motions onto the bilayer normal (9Wiener M.C. White S.H. Biophys. J. 1991; 59: 162-173Abstract Full Text PDF PubMed Scopus (138) Google Scholar, 10Wiener M.C. White S.H. Biophys. J. 1991; 59: 174-185Abstract Full Text PDF PubMed Scopus (107) Google Scholar). The liquid crystallographic structure of an Lα-phase dioleoylphosphatidylcholine (DOPC) bilayer is shown in Fig. 2A (11Wiener M.C. White S.H. Biophys. J. 1992; 61: 434-447Abstract Full Text PDF PubMed Scopus (649) Google Scholar). Three features of this structure are important. First, the widths of the probability densities reveal the great thermal disorder of fluid membranes. Second, the combined thermal thicknesses of the IFs (defined by the distribution of the waters of hydration) is about equal to the 30-Å thickness of the HC. The thermal thickness of a single IF (∼15 Å) can easily accommodate an α-helix parallel to the membrane plane (Fig. 2 B). The common cartoons of bilayers that assign a diminutive thickness to the bilayer IFs are thus misleading. Third, the thermally disordered IFs are highly heterogeneous chemically. As the regions of first contact, the IFs are especially important in the folding and insertion of non-constitutive MPs, such as toxins (12Lindeberg M. Zakharov S.D. Cramer W.A. J. Mol. Biol. 2000; 295: 679-692Crossref PubMed Scopus (58) Google Scholar), and to the activity of surface-binding enzymes, such as phospholipase A2 (13Ghomashchi F. Lin Y. Hixon M.S., Yu, B.-Z. Annand R. Jain M.K. Gelb M.H. Biochemistry. 1998; 37: 6697-6710Crossref PubMed Scopus (52) Google Scholar). But they are also important in shaping MP structure (Fig. 1). A molecule moving from water to the bilayer HC must experience a dramatic variation in environmental polarity over a short distance because of interfacial chemical heterogeneity, as illustrated by theyellow curve of Fig. 2 B (14White S.H. Wimley W.C. Biochim. Biophys. Acta. 1998; 1376: 339-352Crossref PubMed Scopus (455) Google Scholar). An amphipathic helix such as melittin (15Dempsey C.E. Biochim. Biophys. Acta. 1990; 1031: 143-161Crossref PubMed Scopus (785) Google Scholar), represented schematically in Fig. 2 B, locates (16Hristova K. Dempsey C.E. White S.H. Biophys. J. 2001; 80: 801-811Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar) at the midpoint of the steep descent of the polarity gradient. Because the polarity changes over a distance corresponding roughly to helix diameter, peptide-bilayer interaction energies must be very sensitive to polarized helices, such as amphipathic ones. Experimental exploration of the stability of intact MPs is problematic because of their general insolubility. One approach to stability is to "divide and conquer" by studying the membrane interactions of fragments of MPs, i.e. peptides. Because MPs are equilibrium structures, folding and stability can be examined by constructing thermodynamic pathways (3White S.H. Wimley W.C. Annu. Rev. Biophys. Biomol. Struct. 1999; 28: 319-365Crossref PubMed Scopus (1472) Google Scholar) such as those shown in Fig.3. Although these pathways do not mirror the actual biological assembly process of MPs, they are nevertheless useful for guiding biological experiments, because they provide a thermodynamic context within which biological processes must proceed. The four-step model (3White S.H. Wimley W.C. Annu. Rev. Biophys. Biomol. Struct. 1999; 28: 319-365Crossref PubMed Scopus (1472) Google Scholar) of Fig. 3 is a logical combination of an early three-step model of Jacobs and White (17Jacobs R.E. White S.H. Biochemistry. 1989; 28: 3421-3437Crossref PubMed Scopus (425) Google Scholar) and the two-stage model of Popot and Engelman (18Popot J.-L. Gerchman S.-E. Engelman D.M. J. Mol. Biol. 1987; 198: 655-676Crossref PubMed Scopus (243) Google Scholar, 19Popot J.-L. Engelman D.M. Biochemistry. 1990; 29: 4031-4037Crossref PubMed Scopus (820) Google Scholar) in which TM helices are first "established" across the membrane and then assemble into functional structures (helix association). The model summarizes the types of experiments on MP folding now being pursued in several laboratories. In Fig. 3, the free energy reference state is taken as the unfolded protein in an IF. However, this state cannot actually be achieved with MPs because of the solubility problems, nor can it be achieved with small non-constitutive membrane-active peptides, such as melittin, because binding usually induces secondary structure (partitioning-folding coupling). Thus, as is often the case in solution thermodynamics, the reference state must be a virtual one. It can be defined for phosphocholine IFs by means of an experimental interfacial free energy (hydrophobicity) scale (20Wimley W.C. White S.H. Nat. Struct. Biol. 1996; 3: 842-848Crossref PubMed Scopus (1385) Google Scholar) derived from the partitioning into POPC bilayers of tri- and pentapeptides (17Jacobs R.E. White S.H. Biochemistry. 1989; 28: 3421-3437Crossref PubMed Scopus (425) Google Scholar, 20Wimley W.C. White S.H. Nat. Struct. Biol. 1996; 3: 842-848Crossref PubMed Scopus (1385) Google Scholar) that have no secondary structure in the aqueous or interfacial phases. This scale, which includes the peptide bonds as well as the side chains, allows calculation of the virtual free energy of transfer of an unfolded chain into an IF. For peptides that cannot form regular secondary structure, such as the antimicrobial peptide indolicidin (21Ladokhin A.S. Selsted M.E. White S.H. Biochemistry. 1999; 38: 12313-12319Crossref PubMed Scopus (125) Google Scholar), the scale predicts observed free energies of transfer with remarkable accuracy (22Ladokhin A.S. White S.H. J. Mol. Biol. 2001; 309: 543-552Crossref PubMed Scopus (103) Google Scholar). This validates it for the computation of virtual free energies for partitioning into phosphocholine IFs. Similar scales are needed for other lipids and lipid mixtures. The high cost of interfacial partitioning of the peptide bond (20Wimley W.C. White S.H. Nat. Struct. Biol. 1996; 3: 842-848Crossref PubMed Scopus (1385) Google Scholar), 1.2 kcal mol−1, explains the origin of partitioning-folding coupling and also why the interface is a potent catalysis of secondary structure formation. Wimley et al. (23Wimley W.C. Hristova K. Ladokhin A.S. Silvestro L. Axelsen P.H. White S.H. J. Mol. Biol. 1998; 277: 1091-1110Crossref PubMed Scopus (183) Google Scholar) showed for interfacial β-sheet formation that H-bond formation reduces the cost of peptide partitioning by about 0.5 kcal mol−1 per peptide bond. The folding of melittin into an amphipathic α-helix on POPC membranes involves a per residue reduction of about 0.4 kcal mol−1 (24Ladokhin A.S. White S.H. J. Mol. Biol. 1999; 285: 1363-1369Crossref PubMed Scopus (287) Google Scholar). The folding of the antimicrobial peptide magainin on charged bilayers seems to entail a smaller per residue value, about 0.1 kcal mol−1 (25Wieprecht T. Beyermann M. Seelig J. Biochemistry. 1999; 38: 10377-10387Crossref PubMed Scopus (175) Google Scholar). The cumulative effect of these relatively small per residue free energy reductions can be very large when tens or hundreds of residues are involved, as in the assembly of the β-barrel transmembrane domain (26Song L. Hobaugh M.R. Shustak C. Cheley S. Bayley H. Gouaux J.E. Science. 1996; 274: 1859-1866Crossref PubMed Scopus (1927) Google Scholar) of α-hemolysin that buries ∼100 residues in the membrane. Determination of the energetics of TM α-helix insertion, which is critically important for predicting structure, is difficult because non-polar helices tend to aggregate in both the aqueous and interfacial phases (27Wimley W.C. White S.H. Biochemistry. 2000; 39: 4432-4442Crossref PubMed Scopus (128) Google Scholar). Several efforts have been made, with mixed success (27Wimley W.C. White S.H. Biochemistry. 2000; 39: 4432-4442Crossref PubMed Scopus (128) Google Scholar, 28Moll T.S. Thompson T.E. Biochemistry. 1994; 33: 15469-15482Crossref PubMed Scopus (42) Google Scholar, 29Soekarjo M. Eisenhawer M. Kuhn A. Vogel H. Biochemistry. 1996; 35: 1232-1241Crossref PubMed Scopus (59) Google Scholar, 30Bechinger B. J. Mol. Biol. 1996; 263: 768-775Crossref PubMed Scopus (173) Google Scholar, 31Hunt J.F. Rath P. Rothschild K.J. Engelman D.M. Biochemistry. 1997; 36: 15177-15192Crossref PubMed Scopus (205) Google Scholar). Although precise values for the free energy of helix insertion remain to be established, the broad energetic issues are clear (32Roseman M.A. J. Mol. Biol. 1988; 201: 621-625Crossref PubMed Scopus (101) Google Scholar). Computational studies (33Ben-Tal N. Sitkoff D. Topol I.A. Yang A.-S. Burt S.K. Honig B. J. Phys. Chem. B. 1997; 101: 450-457Crossref Scopus (139) Google Scholar, 34Ben-Tal N. Ben-Shaul A. Nicholls A. Honig B. Biophys. J. 1996; 70: 1803-1812Abstract Full Text PDF PubMed Scopus (201) Google Scholar) suggest that the transfer free energy ΔG CONH of a non-H-bonded peptide bond from water to alkane is +6.4 kcal mol−1, compared with only +2.1 kcal mol−1 for the transfer free energy ΔG Hbond of an H-bonded peptide bond. The per residue free energy cost of disrupting H-bonds in a membrane is therefore about 4 kcal mol−1. A 20-amino acid TM helix would cost 80 kcal mol−1 to unfold within a membrane, which explains why unfolded polypeptide chains cannot exist in a transmembrane configuration. Fig. 4illustrates the importance of ΔG Hbond in setting the threshold for transmembrane stability as well as the so-called decision level in hydropathy plots (35White S.H. White S.H. Membrane Protein Structure: Experimental Approaches. Oxford University Press, New York1994: 97-124Crossref Google Scholar). Using the single membrane-spanning helix of glycophorin A (36Segrest J.P. Jackson R.L. Marchesi V.T. Guyer R.B. Terry W. Biochem. Biophys. Res. Commun. 1972; 49: 964-969Crossref PubMed Scopus (87) Google Scholar) as an example, panel A shows that the free energy of transfer of the side chains dramatically favors helix insertion, whereas the transfer cost of the helical backbone dramatically disfavors insertion. Panel B shows that an uncertainty of 0.5 kcal mol−1 in the per residue cost of backbone insertion has a major effect on the interpretation of hydropathy plots and on the establishment of the minimum value of side chain hydrophobicity required for transmembrane helix stability. What is the most likely estimate of ΔG Hbond? The practical number, in the context of Fig. 4 A, is the cost of ΔG glycylhelixtransferring a single glycyl unit of a polyglycine α-helix into the bilayer HC. Electrostatic calculations (34Ben-Tal N. Ben-Shaul A. Nicholls A. Honig B. Biophys. J. 1996; 70: 1803-1812Abstract Full Text PDF PubMed Scopus (201) Google Scholar) and the octanol partitioning study of Wimley et al. (37Wimley W.C. Creamer T.P. White S.H. Biochemistry. 1996; 35: 5109-5124Crossref PubMed Scopus (471) Google Scholar) suggest that ΔG glycylhelix = +1.25 kcal mol−1, which is the basis for ΔG bb in Fig. 4 A. Interestingly, the cost of transferring a random-coil glycyl unit inton-octanol (37Wimley W.C. Creamer T.P. White S.H. Biochemistry. 1996; 35: 5109-5124Crossref PubMed Scopus (471) Google Scholar) is +1.15 kcal mol−1. This suggests that the n-octanol whole-residue hydrophobicity scale (3White S.H. Wimley W.C. Annu. Rev. Biophys. Biomol. Struct. 1999; 28: 319-365Crossref PubMed Scopus (1472) Google Scholar) derived from the partitioning data of Wimley et al. (37Wimley W.C. Creamer T.P. White S.H. Biochemistry. 1996; 35: 5109-5124Crossref PubMed Scopus (471) Google Scholar) may be a good measure of ΔG glycylhelix and therefore useful for identifying α-helical TM segments in hydropathy plots of MPs (3White S.H. Wimley W.C. Annu. Rev. Biophys. Biomol. Struct. 1999; 28: 319-365Crossref PubMed Scopus (1472) Google Scholar). This is borne out by work in progress 2Jayasinghe, S., Hristova, K., and White, S. H., (2001) J. Mol. Biol., in press. using the recently developed MPtopo data base of MPs of known topology (38Jayasinghe S. Hristova K. White S.H. Protein Sci. 2001; 10: 455-458Crossref PubMed Scopus (147) Google Scholar), accessible via the World Wide Web (blanco.biomol.uci.edu/mptopo). The hydrophobic effect is generally considered to be the major driving force for compacting soluble proteins (39Dill K.A. Biochemistry. 1990; 29: 7133-7155Crossref PubMed Scopus (3311) Google Scholar). However, it cannot be the force driving compaction (association) of TM α-helices. Because the hydrophobic effect arises solely from dehydration of a non-polar surface (40Tanford C. The Hydrophobic Effect: Formation of Micelles and Biological Membranes. John Wiley 28: 319-365Crossref PubMed Scopus (1472) Google Scholar and 4Popot J.-L. Engelman D.M. Annu. Rev. Biochem. 2000; 69: 881-922Crossref PubMed Scopus (535) Google Scholar), but why would van der Waals forces be stronger between helices than between helices and lipids? Extensive work (41Lemmon M.A. Flanagan J.M. Hunt J.F. Adair B.D. Bormann B.J. Dempsey C.E. Engelman D.M. J. Biol. Chem. 1992; 267: 7683-7689Abstract Full Text PDF PubMed Google Scholar, 42Lemmon M.A. Treutlein H.R. Adams P.D. Brünger A.T. Engelman D.M. Nat. Struct. Biol. 1994; 1: 157-163Crossref PubMed Scopus (297) Google Scholar, 43MacKenzie K.R. Prestegard J.H. Engelman D.M. Science. 1997; 276: 131-133Crossref PubMed Scopus (869) Google Scholar, 44Fleming K.G. Ackerman A.L. Engelman D.M. J. Mol. Biol. 1997; 272: 266-275Crossref PubMed Scopus (207) Google Scholar, 45MacKenzie K.R. Engelman D.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3583-3590Crossref PubMed Scopus (130) Google Scholar) on dimer formation of glycophorin A in detergents reveals the answer: knob-into-hole packing that allows more efficient packing between helices than between helices and lipids. Tight, knob-into-hole packing has been found to be a general characteristic of helical bundle MPs as well (46Bowie J.U. J. Mol. Biol. 1997; 272: 780-789Crossref PubMed Scopus (279) Google Scholar, 47Langosch D. Heringa J. Proteins. 1998; 31: 150-159Crossref PubMed Scopus (121) Google Scholar). For glycophorin A dimerization, knob-into-hole packing is facilitated by the GXXXG motif, in which the glycines permit close approach of the helices. The substitution of larger residues for glycine prevents the close approach and hence dimerization (41Lemmon M.A. Flanagan J.M. Hunt J.F. Adair B.D. Bormann B.J. Dempsey C.E. Engelman D.M. J. Biol. Chem. 1992; 267: 7683-7689Abstract Full Text PDF PubMed Google Scholar, 44Fleming K.G. Ackerman A.L. Engelman D.M. J. Mol. Biol. 1997; 272: 266-275Crossref PubMed Scopus (207) Google Scholar,45MacKenzie K.R. Engelman D.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3583-3590Crossref PubMed Scopus (130) Google Scholar). The so-called TOX-CAT method (48Russ W.P. Engelman D.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 863-868Crossref PubMed Scopus (323) Google Scholar) has made it possible to sample the amino acid motifs preferred in helix-helix association in membranes by using randomized sequence libraries (49Russ W.P. Engelman D.M. J. Mol. Biol. 2000; 296: 911-919Crossref PubMed Scopus (781) Google Scholar). The GXXXG motif is among a significant number of motifs that permit close packing. A statistical survey of MP sequences disclosed that these motifs are very common in membrane proteins (50Senes A. Gerstein M. Engelman D.M. J. Mol. Biol. 2000; 296: 921-936Crossref PubMed Scopus (512) Google Scholar). Dimerization studies of glycophorin in detergent micelles (44Fleming K.G. Ackerman A.L. Engelman D.M. J. Mol. Biol. 1997; 272: 266-275Crossref PubMed Scopus (207) Google Scholar) do not permit the absolute free energy of association to be determined because of the large free energy changes associated with micelle stability. However, estimates (3White S.H. Wimley W.C. Annu. Rev. Biophys. Biomol. Struct. 1999; 28: 319-365Crossref PubMed Scopus (1472) Google Scholar) suggest 1–5 kcal mol−1 as the free energy cost of separating a helix from a helix bundle within the bilayer environment. The cost of breaking H-bonds within the bilayer HC (above) implies that H-bonding between α-helices could provide a strong stabilizing force for helix association. This is borne out by recent studies of synthetic TM peptides designed to hydrogen bond to one another (51Zhou F.X. Cocco M.J. Russ W.P. Brunger A.T. Engelman D.M. Nat. Struct. Biol. 2000; 7: 154-160Crossref PubMed Scopus (356) Google Scholar, 52Choma C. Gratkowski H. Lear J.D. DeGrado W.F. Nat. Struct. Biol. 2000; 7: 161-166Crossref PubMed Scopus (346) Google Scholar). Interhelical H-bonds, however, are not common in MPs (reviewed in Ref. 3White S.H. Wimley W.C. Annu. Rev. Biophys. Biomol. Struct. 1999; 28: 319-365Crossref PubMed Scopus (1472) Google Scholar). Indeed, lacking the specificity of knobs-into-hole packing, they could be hazardous because of their tendency to cause promiscuous aggregation (4Popot J.-L. Engelman D.M. Annu. Rev. Biochem. 2000; 69: 881-922Crossref PubMed Scopus (535) Google Scholar). However, they are probably important in the association of transmembrane signaling proteins (53Smith S.O. Smith C.S. Bormann B.J. Nat. Struct. Biol. 1996; 3: 252-258Crossref PubMed Scopus (153) Google Scholar). As for soluble proteins, the ultimate solution to the problem of predicting three-dimensional structure of MPs from sequence will come from a deep quantitative understanding of the energetics of protein folding. The experimental approaches described above lead in that direction. At a simple level, the prediction of MP topology is fairly easy and reliable because of the high hydrophobicity of TM helices. Such sequence segments are generally apparent in hydropathy analysis (Fig. 4 B), which is now a prediction (reviewed in Ref. S.H. White S.H. Membrane Protein Structure: Experimental Approaches. Oxford University Press, New York1994: 97-124Crossref Google Scholar). However, the of the resulting the hydrophobicity scale and are side chain only An using the MPtopo data base (38Jayasinghe S. Hristova K. White S.H. Protein Sci. 2001; 10: 455-458Crossref PubMed Scopus (147) Google Scholar) reveals that side chain only scales TM segments because of the of ΔG bb for illustrated by B. The whole-residue hydrophobicity scale of White and Wimley (3White S.H. Wimley W.C. Annu. Rev. Biophys. Biomol. Struct. 1999; 28: 319-365Crossref PubMed Scopus (1472) Google Scholar), which ΔG bb into reduces Membrane Protein is a hydropathy analysis using this scale The into prediction of of MP structure and such as the so-called J. Mol. Biol. 1992; PubMed Scopus Google Scholar, 1994; 10: Google Scholar) or secondary structure C. A.S. S. Protein Sci. 2001; 10: PubMed Scopus Google Scholar), can the of topology prediction that in on of MP sequences with significant to a sequence of can also accuracy F. P. B. Biochem. Sci. 1995; Full Text PDF PubMed Scopus Google Scholar, B. R. P. C. Protein Sci. 1995; PubMed Scopus Google Scholar, Rev. 1998; PubMed Google Scholar, Simon J. Mol. Biol. 1998; PubMed Scopus Google Scholar, K.A. J. T. F. R. D. C. of the on for Press, Scholar). progress has been made the in understanding the physical principles MP structure and stability. great importance is the number of MPs structures have been determined to high resolution is at structures have now been and are helical or An important is motifs will they may they would have to include H-bonded peptide bonds in the transmembrane One is the motif F. Science. PubMed Scopus Google Scholar). A significant of MPs, such as C. M. H. H. 2000; PubMed Scopus Google Scholar), is large This means that the prediction of MP structure will as well success in predicting the structure of soluble proteins. not in prediction is the of which are common in large MPs. information about the assembly of MPs by the translocon may in into structure New are also likely to from understanding of the of lipids in MP folding (reviewed in Refs. M. W. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar and F. B. Biochem. J. 2000; PubMed Scopus Google Scholar). a more understanding of in will how membrane interfaces shape protein structure. importance are the interactions of residues Biochem. Sci. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar) and charged residues D. A. Honig B. S. Simon S.A. in Press, New Scholar), and how hydrophobic and interactions to proteins at interfaces (22Ladokhin A.S. White S.H. J. Mol. Biol. 2001; 309: 543-552Crossref PubMed Scopus (103) Google Scholar). We for We are especially to the of Wimley to the work of and to of the in this