Self-association of Transmembrane α-Helices in Model Membranes

Interactions between transmembrane helices play a key role in almost all cellular processes involving membrane proteins. We have investigated helix-helix interactions in lipid bilayers with synthetic tryptophan-flanked peptides that mimic the membrane spanning parts of membrane proteins. The peptides were functionalized with pyrene to allow the self-association of the helices to be monitored by pyrene fluorescence and Trp-pyrene fluorescence resonance energy transfer (FRET). Specific labeling of peptides at either their N or C terminus has shown that helix-helix association occurs almost exclusively between antiparallel helices. Furthermore, computer modeling suggested that antiparallel association arises primarily from the electrostatic interactions between α-helix backbone atoms. We propose that such interactions may provide a force for the preferentially antiparallel association of helices in polytopic membrane proteins. Helix-helix association was also found to depend on the lipid environment. In bilayers of dioleoylphosphatidylcholine, in which the hydrophobic length of the peptides approximately matched the bilayer thickness, association between the helices was found to require peptide/lipid ratios exceeding 1/25. Self-association of the helices was promoted by either increasing or decreasing the bilayer thickness, and by adding cholesterol. These results indicate that helix-helix association in membrane proteins can be promoted by unfavorable protein-lipid interactions. Interactions between transmembrane helices play a key role in almost all cellular processes involving membrane proteins. We have investigated helix-helix interactions in lipid bilayers with synthetic tryptophan-flanked peptides that mimic the membrane spanning parts of membrane proteins. The peptides were functionalized with pyrene to allow the self-association of the helices to be monitored by pyrene fluorescence and Trp-pyrene fluorescence resonance energy transfer (FRET). Specific labeling of peptides at either their N or C terminus has shown that helix-helix association occurs almost exclusively between antiparallel helices. Furthermore, computer modeling suggested that antiparallel association arises primarily from the electrostatic interactions between α-helix backbone atoms. We propose that such interactions may provide a force for the preferentially antiparallel association of helices in polytopic membrane proteins. Helix-helix association was also found to depend on the lipid environment. In bilayers of dioleoylphosphatidylcholine, in which the hydrophobic length of the peptides approximately matched the bilayer thickness, association between the helices was found to require peptide/lipid ratios exceeding 1/25. Self-association of the helices was promoted by either increasing or decreasing the bilayer thickness, and by adding cholesterol. These results indicate that helix-helix association in membrane proteins can be promoted by unfavorable protein-lipid interactions. Most membrane proteins have one or more hydrophobic segments that span the membrane in an α-helical conformation. Interactions between these transmembrane (TM) 4The abbreviations used are:TMtransmembraneFRETfluorescence resonance energy transferr.m.s.root mean squarePCphosphatidylcholineHPLChigh performance liquid chromatographyE/Mexcimer/monomer ratio 4The abbreviations used are:TMtransmembraneFRETfluorescence resonance energy transferr.m.s.root mean squarePCphosphatidylcholineHPLChigh performance liquid chromatographyE/Mexcimer/monomer ratio helices are important for determining the structure of multispanning membrane proteins and for assembly of membrane proteins into oligomeric structures (1Arkin I.T. Biochim. Biophys. Acta. 2002; 1565: 347-363Crossref PubMed Scopus (48) Google Scholar, 2Chamberlain A. Faham S. Yohannan S. Bowie J. Rees D.C. Advances in Protein Chemistry. 63. Academic Press, Amsterdam2003: 19-46Google Scholar, 3Engelman D.M. Chen Y. Chin C.N. Curran A.R. Dixon A.M. Dupuy A.D. Lee A.S. Lehnert U. Matthews E.E. Reshetnyak Y.K. Senes A. Popot J.L. FEBS Lett. 2003; 555: 122-125Crossref PubMed Scopus (247) Google Scholar, 4Langosch D. Lindner E. Gurezka R. IUBMB Life. 2002; 54: 109-113Crossref PubMed Scopus (15) Google Scholar). Several factors are thought to be responsible for the association of helices in membrane proteins, including surface complementarity, the presence of polar residues in the transmembrane region (5Zhou F.X. Merianos H.J. Brunger A.T. Engelman D.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2250-2255Crossref PubMed Scopus (314) Google Scholar, 6Choma C. Gratkowski H. Lear J.D. DeGrado W.F. Nat. Struct. Biol. 2000; 7: 161-166Crossref PubMed Scopus (341) Google Scholar, 7Johnson R.M. Heslop C.L. Deber C.M. Biochemistry. 2004; 43: 14361-14369Crossref PubMed Scopus (31) Google Scholar), and certain specific motifs such as the well known GXXXG pattern (8MacKenzie K.R. Prestegard J.H. Engelman D.M. Science. 1997; 276: 131-133Crossref PubMed Scopus (865) Google Scholar, 9Senes A. Ubarretxena-Belandia I. Engelman D.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9056-9061Crossref PubMed Scopus (432) Google Scholar). It is likely that several of these factors act in concert to determine the final folded structure, or the association of monomers to form an oligomer. transmembrane fluorescence resonance energy transfer root mean square phosphatidylcholine high performance liquid chromatography excimer/monomer ratio transmembrane fluorescence resonance energy transfer root mean square phosphatidylcholine high performance liquid chromatography excimer/monomer ratio In addition to helix-helix interactions, interactions between the helices and surrounding lipids also play a role in the organization and assembly of TM helices. For example, even when helices do not exhibit any tendency to undergo specific association (10Lee A.G. Biochim. Biophys. Acta. 2004; 1666: 62-87Crossref PubMed Scopus (918) Google Scholar, 11Killian J.A. Biochim. Biophys. Acta. 1998; 1376: 401-416Crossref PubMed Scopus (510) Google Scholar, 12Mall S. Broadbridge R. Sharma R.P. East J.M. Lee A.G. Biochemistry. 2001; 40: 12379-12386Crossref PubMed Scopus (70) Google Scholar), helix-helix association could still occur as a result of poor packing between the lipids and helices, or from a favorable change in entropy resulting from the release of helix-bound lipids upon helix association. In these cases, helix association is primarily driven by lipid-protein interactions rather than strongly favorable protein-protein interactions. It is likely that in real membrane proteins the driving forces for folding involve both types of interaction, whether or not specific protein-protein recognition motifs are present. One property of a protein-lipid system that is known to affect helix-helix association is the extent of matching between the hydrophobic length of the helices and the hydrophobic thickness of the lipid bilayer. In the case of hydrophobic mismatch, helix-helix interactions may be promoted because of relatively unfavorable lipid-helix interactions. It was previously shown with Lys-flanked TM peptides that hydrophobic mismatch does promote self-association, both when a helix-helix recognition motif is present (13Orzáez M. Lukovic D. Abad C. Pérez-Payá E. Mingarro I. FEBS Lett. 2005; 579: 1633-1638Crossref PubMed Scopus (32) Google Scholar), and in the absence of such a motif (14Webb R.J. East J.M. Sharma R.P. Lee A.G. Biochemistry. 1998; 37: 673-679Crossref PubMed Scopus (131) Google Scholar, 15Ren J.H. Lew S. Wang J.Y. London E. Biochemistry. 1999; 38: 5905-5912Crossref PubMed Scopus (114) Google Scholar). However, it is still not clear whether or not helix-helix association can be considered a general response of TM helices to mismatch. This is because many other responses to hydrophobic mismatch can also occur (reviewed in Refs. 16Killian J.A. FEBS Lett. 2003; 555: 134-138Crossref PubMed Scopus (134) Google Scholar and 17de Planque M.R.R. Killian J.A. Mol. Membr. Biol. 2003; 20: 271-284Crossref PubMed Scopus (255) Google Scholar), such as ordering/disordering of the lipid acyl chains, alterations in helix tilt angle, adaptations of the peptide backbone, and because it has been shown that the type and extent of the responses that occur depend on the composition of the TM helix. For example, Trp-flanked peptides, which were designed to mimic the membrane spanning parts of intrinsic membrane proteins, showed very different responses to hydrophobic mismatch than analogous Lys-flanked peptides (14Webb R.J. East J.M. Sharma R.P. Lee A.G. Biochemistry. 1998; 37: 673-679Crossref PubMed Scopus (131) Google Scholar, 15Ren J.H. Lew S. Wang J.Y. London E. Biochemistry. 1999; 38: 5905-5912Crossref PubMed Scopus (114) Google Scholar). The aims of the present study are to establish whether or not increased association is a general property of transmembrane segments under conditions of hydrophobic mismatch, and to understand the molecular details of any oligomers that are formed. We investigated the association between Trp-flanked peptides that were designed to mimic the transmembrane segments of real membrane proteins, without specific helix-helix recognition motifs. These model peptides allowed us to focus on nonspecific forces involved in membrane protein structure and stability. The peptides were functionalized with pyrene to allow monitoring of association by pyrene fluorescence and Trp-pyrene FRET. By labeling peptides at either their N or C terminus, we show that helix-helix association occurs almost exclusively between antiparallel helices. We also show that hydrophobic mismatch promotes helix-helix association. Our theoretical models show that the antiparallel association of helices is promoted by favorable electrostatic interactions between α-helix backbone dipole moments. These results contribute to our understanding of the role of protein-protein and protein-lipid interactions in determining the association of transmembrane segments in membrane proteins. The implications of these findings for assembly and stability of membrane proteins and membrane protein complexes are also discussed. Materials—1,2-Dioleoyl-sn-glycero-3-phosphocholine (C18:1c-PC), 1,2-dimyristoleoyl-sn-glycero-3-phosphocholine (C14:1c-PC), 1,2-dierucoyl-sn-glycero-3-phosphocholine (C22:1c-PC), and cholesterol were obtained from Avanti Polar Lipids (Alabaster, AL). The pyrene-labeled peptides pyrN-WALP23 (Ac-C(pyrene)-GWW(LA)8LWWA-amide) and pyrC-WALP23 (Ac-GWW(LA)8LWWGC(pyrene)-amide) were synthesized as described earlier (18Sparr E. Ganchev D.N. Snel M.M.E. Ridder A.N.J.A. Kroon-Batenburg L.M.J. Chupin V. Rijkers D.T.S. Killian J.A. de Kruijff B. Biochemistry. 2005; 44: 2-10Crossref PubMed Scopus (17) Google Scholar). The fluorescent probe N-(1-pyrene)maleimide was obtained from Molecular Probes Europe BV (Leiden, The Netherlands). The peptides were purified by high performance liquid chromatography (HPLC). Their identity was confirmed by mass spectrometry and their purity was established by analytical HPLC to be better than 95%. Milli-Q water was used for all experiments. Sample Preparation—Fluorescence experiments were performed on multilamellar vesicles with peptide/lipid ratios between 1/10 and 1/3000. The lipids were dissolved in either chloroform/methanol (1/1) or chloroform, and the peptides were dissolved in 2,2,2-trifluoroethanol. The concentration of WALP in the stock solution was determined from the absorbance at 280 nm using an extinction coefficient of 21,300 m-1 cm-1. Phospholipids were quantified according to Rouser et al. (19Rouser G. Fleisher S. A. PubMed Scopus Google Scholar). were into lipid vesicles as described Planque M.R.R. D. de Kruijff B. Killian J.A. J. Biol. 1999; PubMed Scopus Google Scholar). of peptide and lipids were in the peptide/lipid The was by under a of and the peptide/lipid was under The were in Milli-Q water to a final peptide concentration of and the were by The were to of The resulting multilamellar vesicles were at confirmed that the WALP peptides were to form TM helices in lipid experiments were performed with an C were in a The was at a water with In the absorbance of was at the of and on a These were used to the in Google Scholar). The fluorescence of pyrene was with between and nm with an of nm The between and pyrene was determined from the of were between and nm with an of 280 nm In both and pyrene were present in the that the both from energy transfer and from energy The from the was from obtained for vesicles with very peptide In these the was and the could be considered The in study are as the ratio of the to the is energy were by of which an of by a H. A. Lett. 2001; Scopus Google Scholar, J. Sci. 2004; 44: PubMed Scopus Google Scholar). In the modeling the peptides are to be α-helical J. Sci. 2004; 44: PubMed Scopus Google Scholar), and the of the and pyrene is from the The from the at the helix backbone to the of of the and were to be and a model for peptides into a lipid a square region of a bilayer peptides was The peptides were on the of a with the between to Lipids were not in the their and the peptide/lipid ratio were used to the final of the were with the helix of the peptide either or antiparallel to the of the For in the of energy transfer to one of the is by the is the between the and in the and is the which was to in Google Scholar). The all the in the The mean for all the energy transfer in the The protein and were and to the final and of were with a modeling M. Brunger A.T. PubMed Scopus Google Scholar), using the force and previously described J.L. Biochemistry. 2004; 43: PubMed Scopus Google Scholar). We performed different modeling in which the peptides were either or antiparallel to and in which the helix dipole were either or The was by the of the on all and backbone on one helix. This backbone not the structure of helices The modeling were and the the and the the dipole and helix dipole that were from structure was a of nm from of the helices their were used to structures resulting in a of structures for structures resulting from were to than structures for In all cases, the of the helix or by the of both in the or to This was to electrostatic interactions between the which be by water or involved in interactions with the lipid and helix-helix were using the J. Struct. Biol. 2002; PubMed Scopus Google Scholar), We the for to in the helix-helix by by The indicate the of residues of a in the structure that one or more with an than a determined with of the resulting by a in of model and were a were on angle, helix-helix and the of as described These were with a of on For the J. B. Proc. 2004; was used with and the on The was to to at for more than were and on the root mean square of the of all between using the Protein PubMed Scopus Google and an of of the to the structures for of the helices structures for was also using the with This was as a the described as by investigated the interactions between Trp-flanked TM by using pyrene-labeled WALP The pyrene a fluorescence with at between and pyrene are in with form an which a fluorescence at a of The helix-helix was in multilamellar vesicles of and the peptide pyrN-WALP23 at peptide/lipid ratios from to the pyrene showed on the peptide/lipid the was and could not even be at high peptide This that is between peptides that are to However, interactions between helices antiparallel with to other are not with we WALP in the one at the N terminus as described and one at the C terminus and the pyrene for with a peptide/lipid ratio of The pyrene for pyrC-WALP23 was to than that of pyrN-WALP23 that the C terminus of the peptide in a more hydrophobic in Google in the lipid bilayer. This is with from mass spectrometry J. Killian J.A. Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google Scholar), which showed that the C terminus is more from than the N terminus, and from that the peptide is with to the of the membrane J. 2005; PubMed Scopus Google Scholar). was not when one type of these peptides was when pyrN-WALP23 and pyrC-WALP23 were both was was obtained for the of pyrN-WALP23 and pyrC-WALP23 In it is that these results are because of more favorable by pyrene in antiparallel peptides than in However, is because we have previously shown that also occurs in peptides under certain conditions (18Sparr E. Ganchev D.N. Snel M.M.E. Ridder A.N.J.A. Kroon-Batenburg L.M.J. Chupin V. Rijkers D.T.S. Killian J.A. de Kruijff B. Biochemistry. 2005; 44: 2-10Crossref PubMed Scopus (17) Google Scholar). the of our is that is between antiparallel helices. pyrene fluorescence were performed for with peptide/lipid from to The between the excimer/monomer ratio and the peptide concentration is shown in was at high peptide ratios and when both types of peptides were present. The of hydrophobic mismatch on helix-helix interactions was investigated by the pyrene fluorescence for peptides in vesicles of hydrophobic or hydrophobic to obtained for the pyrene obtained for a peptide/lipid ratio of 1/25. We an in when was hydrophobic mismatch between the peptides and the lipid bilayer. was also at peptide than in the matching with an at a peptide/lipid ratio of This that peptide is promoted by hydrophobic mismatch. was between the of and mismatch. when both types of peptides were a organization of the peptides of the extent of hydrophobic mismatch. increased peptide association was when WALP peptides were in bilayers that cholesterol This can be to the increased hydrophobic mismatch because of of the as well as the increased lipid acyl and the phosphatidylcholine in the presence of cholesterol J.H. Biochemistry. PubMed Scopus Google Scholar). as by between helices was also investigated by between and both of which are present in the WALP The of increased at high peptide These experiments were not to be to the antiparallel or of the helices, as the peptides are with on both results were obtained when one type of pyrN-WALP23 or was or when a of peptides was present not we can that the labeling does not interactions in a and that helix association is not promoted by favorable pyrene interactions in one or the The was also for bilayers of different thickness was more when the peptides were in bilayers of and with when the peptides were present in vesicles of This is with the results of our pyrene fluorescence Our that the with increasing peptide/lipid ratios a decreasing between the and the However, the be to whether is because of association between the peptides, or a of the increased of peptides in the bilayer. we performed a theoretical of the the was for with a of peptides and intrinsic association. The show a in the case of the that the peptides are in the bilayer. However, the real the was found to be of the bilayer thickness not the between the and the in the of hydrophobic mismatch that peptide is promoted when is hydrophobic mismatch between the peptide and the This in peptide is at peptide/lipid ratios peptide the between the and of peptides in the even in the case of hydrophobic mismatch. Furthermore, both the and the show that the is the whether the peptide is at the N or the C and investigated the and the molecular of these with models of both antiparallel and with both and backbone dipole and in the models on the of the helix not on whether the helices were or in and antiparallel promoted packing and a of a The identity of residues involved in also showed a on the helix dipole in a of than antiparallel and the between of by in a the and the of showed that antiparallel dipole and in more than and antiparallel well packing motifs were a of involving residues and of one and and of the other by at either a involving residues and of one and and of the other and association in which all residues one of helix are C and with the helices almost or of these motifs were when the helix backbone were antiparallel to one and of the packing of the helices. and not to any packing and these models in very without the length of the helices and These results indicate that the antiparallel association of helices is a result of favorable electrostatic interactions between the α-helix backbone dipole in the antiparallel between organization of helices is to protein association and protein This can be by relatively and specific interactions including the of and favorable and electrostatic interactions between residues (5Zhou F.X. Merianos H.J. Brunger A.T. Engelman D.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2250-2255Crossref PubMed Scopus (314) Google Scholar, 9Senes A. Ubarretxena-Belandia I. Engelman D.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9056-9061Crossref PubMed Scopus (432) Google Scholar, S. London E. Biochemistry. 2003; PubMed Scopus Google Scholar). In the investigated of these forces are and we that the organization of the peptides is determined by relatively interactions between the helices and unfavorable packing interactions between the peptides and This in be a for the of helices in polytopic membrane proteins, in many specific recognition motifs be However, even in TM helices do such recognition the nonspecific interactions still contribute to the energy of association between the helices. The WALP peptides are in liquid lipid bilayers as J.A. FEBS Lett. 2003; 555: 134-138Crossref PubMed Scopus (134) Google Scholar). the pyrene fluorescence experiments we can that association almost exclusively occurs between peptides that are antiparallel with to This is for all different peptide and all lipid of antiparallel has also been for the TM on experiments Y. S. H. H. M. S. Y. Biochemistry. 2002; PubMed Scopus Google Scholar), that antiparallel association is a property for these types of TM be the for antiparallel arises from the that the peptide have a PubMed Scopus Google Scholar, Biophys. PubMed Scopus Google Scholar, Biophys. Mol. Biol. PubMed Scopus Google Scholar). The and of dipole is still a of with that the dipole in proteins is a to the J. H. A. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). One theoretical of helix association using a bilayer suggested that interactions are because of and of the helix B. Biophys. J. PubMed Scopus Google Scholar). Our is that these interactions be relatively are to antiparallel association in the absence of specific helix association The of of the helix may also be in helices are to be in by high peptide concentration or the of a polytopic membrane This has important implications for understanding the folding and association of proteins in of helices are in known structures of membrane proteins M. D. B. Biophys. J. 2004; PubMed Scopus Google Scholar, J. Mol. Biol. 1997; PubMed Scopus Google Scholar). Our computer models showed that antiparallel peptides form well peptides are and have a tendency to form These are on the backbone dipole not on the packing of the helices, which us to the of a packing between WALP helices. This is also suggested by the of the WALP packing motifs. it is not to determine the between these in a membrane using that the involving and is the This modeling was in However, the of to transmembrane proteins such as Engelman D.M. Brunger A.T. Biochemistry. PubMed Scopus Google Scholar), and the of the results with the that the used are to specific The peptides in system form antiparallel rather than involve both and antiparallel interactions between helices, which are not in our experiments. rather than or has also been for or Lys-flanked peptide in liquid bilayers S. Broadbridge R. Sharma R.P. East J.M. Lee A.G. Biochemistry. 2001; 40: 12379-12386Crossref PubMed Scopus (70) Google Scholar). This is also in with which that WALP and peptides are present as monomers or even at high peptide/lipid ratios Planque M.R.R. D. de Kruijff B. Killian J.A. J. Biol. 1999; PubMed Scopus Google Scholar, Planque M.R.R. H. D. Killian J.A. Biochemistry. 1998; 37: PubMed Scopus Google Scholar, R. A. Biochemistry. 1998; 37: PubMed Scopus Google Scholar). Our results have implications for the folding and assembly of membrane proteins. of polytopic membrane proteins interactions between both and antiparallel helix and it can be that the favorable antiparallel packing of helices to the structure of proteins and to their stability. these interactions to be relatively are to antiparallel association association when hydrophobic mismatch or high peptide concentration between This may be analogous to the high concentration of helices by the in a polytopic membrane and may the of proteins. In self-association of membrane proteins interactions between helices that are in a For such specific recognition motifs are to and have been (5Zhou F.X. Merianos H.J. Brunger A.T. Engelman D.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2250-2255Crossref PubMed Scopus (314) Google Scholar, 6Choma C. Gratkowski H. Lear J.D. DeGrado W.F. Nat. Struct. Biol. 2000; 7: 161-166Crossref PubMed Scopus (341) Google Scholar, Engelman D.M. Brunger A.T. Biochemistry. PubMed Scopus Google Scholar). This may be to the favorable packing of helices. the and theoretical of the on helix-helix interactions in the matching we that the WALP peptides have intrinsic tendency to in liquid lipid at high peptide the between helix dipole is not to of the helices in the bilayer. However, in is or hydrophobic mismatch between the peptides and the either because of increased length of the lipids or the of peptide association is In these interactions more favorable than interactions. by hydrophobic mismatch has previously been for Lys-flanked by fluorescence J.H. Lew S. Wang J.Y. London E. Biochemistry. 1999; 38: 5905-5912Crossref PubMed Scopus (114) Google Scholar, S. Broadbridge R. Sharma R.P. Lee A.G. East J.M. Biochemistry. 2000; PubMed Scopus Google Scholar). it to be a general property of However, the Lys-flanked peptides to have a tendency to at mismatch and form oligomeric than the WALP peptides Planque M.R.R. Killian J.A. Mol. Membr. Biol. 2003; 20: 271-284Crossref PubMed Scopus (255) Google Scholar). for be that residues are more and may better of the peptides than the more In any the molecular details of helices are important in the of the lipid in very by hydrophobic mismatch and the presence of cholesterol is likely to play a role in the of membrane proteins, for example, in processes of proteins is to into J. Sci. 2005; PubMed Scopus Google Scholar, J. 2003; 44: PubMed Scopus Google Scholar). of the high cholesterol and the of the in these are to be than the surrounding in which the proteins be present as favorable interactions between the lipids and the proteins may to the form into these association is a key in many cellular processes involving folding and assembly of membrane proteins. The extent of helix-helix association and the of association on all of which contribute to the energy of helix association and to the folding and of membrane proteins and protein Our study that such factors are the favorable interactions between antiparallel helices, and the between lipid-protein and protein-protein interactions. with

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