Synthetic peptides as models for intrinsic membrane proteins
FEBS Letters 555 (2003) 134^138 FEBS 27772 Minireview Synthetic peptides as models for intrinsic membrane proteins J. Antoinette KillianÃ Department of Biochemistry of Membranes, Center for Biomembranes and Lipid Enzymology, Institute of Biomembranes, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands Received 25 August 2003; accepted 1 September 2003 First published online 15 October 2003 Edited by Gunnar von Heijne, Jan Rydstrom and Peter Brzezinski « and the hydrophobic thickness of the bilayer. How can aAbstract There are many ways in which lipids can modulatethe activity of membrane proteins. Simply a change in hydro- hydrophobic mismatch in£uence properties of membrane pro-phobic thickness of the lipid bilayer, for example, already can teins? When the transmembrane segments are too long tohave various consequences for membrane protein organization span the hydrophobic bilayer thickness (positive mismatch),and hence for activity. By using synthetic transmembrane pep- hydrophobic side chains may stick out and thus become ex-tides, it could be established that these consequences include posed to a polar environment, which will be energetically un-peptide oligomerization, tilt of transmembrane segments, and favorable. To reduce this e¡ective mismatch, the system mayreorientation of side chains, depending on the speci¢c properties respond in several ways, as illustrated in Fig. 1. The proteinsof the peptides and lipids used. The results illustrate the poten- may adapt (i) by forming oligomers, thus shielding the ex-tial of the use of synthetic model peptides to establish general posed groups from the more polar environment, (ii) by chang-principles that govern interactions between membrane proteins ing their backbone conformation, (iii) by tilting away from theand surrounding lipids.ß 2003 Federation of European Biochemical Societies. Pub- bilayer normal, thereby reducing their e¡ective length, or (iv)lished by Elsevier B.V. All rights reserved. by changing the orientation of the side chains near the lipid/ water interface region. Similar responses, except tilting, mayKey words: Transmembrane peptide; K-Helix; occur in the case of a negative mismatch, when the transmem-Hydrophobic mismatch; Interfacial anchoring; Snorkeling; brane segments are relatively short. Also lipids may adapt toTryptophan reduce the e¡ective mismatch, either by stretching or disorder- ing their acyl chains, or by adapting their macroscopic orga- nization. Finally, in mixtures of lipids, the proteins might1. Introduction preferentially surround themselves by best-matching lipids, which might lead to biologically important processes such as Most membrane proteins have one or more hydrophobic sorting.segments that span the membrane in an K-helical con¢gura- Although the situation of hydrophobic mismatch clearly istion. The proteins can interact with surrounding lipids in the unfavorable, also the various mismatch responses may be en-membrane via a range of interactions, including hydrophobic ergetically costly. To gain insight into the energy costs ofinteractions with the hydrophobic membrane interior and individual mismatch responses, one should investigate underelectrostatic interactions, hydrogen bonding and dipolar inter- what conditions they occur, and to what extent they occur.actions in the lipid/water interfacial region. Not surprisingly, This can only be accomplished by using systematic ap-the activity of many membrane proteins has been shown to proaches. Synthetic model peptides are ideally suited for thisdepend on the lipid environment and/or to require speci¢c type of research, because they allow systematic variation oflipids (reviewed in ref. ). To understand how lipids modu- both the hydrophobic length and the composition of mem-late protein activity, knowledge is required of how lipids can brane spanning segments. Typical peptide families that havein£uence structural properties of membrane proteins. This been used consist of a hydrophobic region of polyLeu orminireview will focus on the in£uence of bilayer thickness alternating Leu and Ala with variable length (reviewed inon membrane protein structure and organization and the in- ref. . Most peptides are either £anked with lysine residuessights that have been obtained by using synthetic peptides as to inhibit peptide aggregation and to better secure a stablemodels for membrane proteins. transmembrane orientation [4^7], or with Trp, as a residue E¡ects of variation of bilayer thickness have been observed that frequently £anks transmembrane domains in intrinsicon the functional activity of various membrane proteins upon membrane proteins [8,9]. Examples of these peptide familiesreconstitution in lipid bilayers (reviewed in ref.  and have are Trp-£anked WALP peptides and Lys-£anked KALP pep-been attributed to a mismatch between the hydrophobic tides (Fig. 2). The results obtained with these peptides illus-length of the membrane spanning segments of the protein trate both the huge variety in mismatch responses and the important role of £anking residues. Most of the mismatch responses have been investigated in*Fax: (31)-30-253 39 69.E-mail address: email@example.com (J.A. Killian). single-lipid systems. More complex mixtures of lipids are re- quired only to investigate preferential interactions betweenAbbreviations: P/L, peptide/lipid molar ratio peptides and lipids, for example to determine whether and0014-5793 / 03 / $22.00 ß 2003 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.doi:10.1016/S0014-5793(03)01154-2
J.A. Killian/FEBS Letters 555 (2003) 134^138 135 only a small amount of peptide can be stably incorporated, FTIR measurements show that the peaks in the amide I re- gion remain at the same position and do not change their lineshape, demonstrating that the peptides indeed form very stable K-helices . 2.3. Tilt By tilting away from the bilayer normal peptides could reduce their e¡ective length, thereby compensating for a pos- itive mismatch. Such tilting can indeed occur, as was observed by using solid state 15 N NMR for Lys-£anked peptides .Fig. 1. Schematic illustration of various mismatch responses that Although in these experiments the absolute values of the tiltcan occur under conditions of positive mismatch. For details see the angle could not be quanti¢ed, the data indicate that the in-text. crease in tilt angle with increasing mismatch is rather small and cannot be su⁄cient to compensate for the mismatch. Interestingly, it is possible that also the extent of tilting mayto what extent molecular sorting occurs. It is important here be dependent on the £anking residues, because FTIR andto realize that in biological membranes sorting is not per se solid state 2 H NMR experiments indicated no or only a smallthe energetically most favorable response to mismatch, be- tilt for WALP peptides [9,12], even under conditions of con-cause, just like other mismatch responses, it has an energy siderable mismatch . Clearly, there must be an energy costcost to it, in this case related to a loss of entropy. of tilting, which may be due to a disturbance of the packing of This review will be focussed on the various mismatch re- lipids adjacent to a tilted helix, and/or to an unfavorablesponses that have been studied for model peptides under con- orientation of the side chains of a tilted helix near the lipid/ditions of positive and negative mismatch in single-lipid sys- water interface.tems. 2.4. Side chain reorientation and interaction of £anking2. Consequences of positive mismatch for protein and lipid residues with interfacial region organization Also a change in the orientation of side chains near the lipid/water interface may a¡ect the e¡ective hydrophobic2.1. Aggregation length of a peptide. For example, positioning of the backbone When the hydrophobic length of a peptide exceeds the bi- CK of a Leu in the more polar region, while pointing the sidelayer thickness, this may result in a tendency of the peptides chain towards the membrane interior, may result in a decreaseto aggregate. This may be expressed as an increase in peptide^ of the e¡ective hydrophobic length. For Lys, with its aliphaticpeptide interactions upon increasing the extent of mismatch. chain and positively charged end, it is di⁄cult to visualizeSuch a response indeed has been observed for Lys-£anked how in the case of positive mismatch reorientation of thispeptides by £uorescence measurements [6,10]. Upon increas- side chain could result in a decrease of the hydrophobicing the peptide/lipid molar ratio (P/L) and/or the extent of length, and this is considered unlikely. In contrast to Leumismatch, the tendency to aggregate may ultimately lead to and Lys, Trp is relatively rigid. However, it is also ratherthe formation of macroscopic aggregates. For WALP peptides large, and therefore the precise orientation of this side chain,it was shown that under such conditions peptide-enriched ag- in particular the position of the indole NH, can be expected togregates are formed, that can be separated from a peptide- signi¢cantly in£uence the e¡ective peptide length.depleted bilayer by sucrose density gradient centrifugation . Additional ‘mismatch’ parameters could result from prefer-In line with these results, Fourier transform infrared spectros- ential interactions of speci¢c side chains in the membrane/copy (FTIR) measurements  indicated that the amount of water interface region. The e¡ective length of the peptidepeptide that can be stably incorporated in a lipid bilayer de- would then also be dependent on the exact position at thepends on the extent of mismatch: the larger the mismatch, theless peptide can be incorporated. Interestingly, it was foundthat KALP peptides can more easily be incorporated in lipidbilayers at a large extent of positive mismatch than WALPpeptides , providing a ¢rst indication that the £ankingresidues may play an important role in determining conse-quences of mismatch.2.2. Backbone conformation Theoretically also the peptide backbone could adapt to apositive mismatch, by decreasing its pitch and forming, e.g., aZ-helix. However, this does not seem to be a favorable re-sponse, because WALP peptides show a stable K-helical con-formation that is independent of the extent of mismatch, asobserved by FTIR measurements  and by solid state 2 Hnuclear magnetic resonance (NMR) experiments . Evenunder conditions of a considerable positive mismatch, when Fig. 2. General structure of WALP peptides and KALP peptides.
136 J.A. Killian/FEBS Letters 555 (2003) 134^138interface where this preferred ‘anchoring’ interaction takes geometry of the HII phase, are much more disordered than inplace, and the energy cost of mismatch would be dependent a bilayer and therefore have a reduced hydrophobic length.on the strength of this interaction. Trp has been shown to Also in this model the peptides form large linear aggre-have a preferred interaction with the interfacial region [14^ gates.16]. This results in a resistance of Trp against displacement Non-lamellar phases can be induced both by WALP andfrom the interface towards the aqueous phase upon inducing a KALP peptides under conditions of negative mismatch where-positive mismatch, as shown for WALP peptides by mass by the type of non-lamellar phase is determined by the precisespectrometry in combination with 1 H^2 H exchange  and extent of negative mismatch . An important implication ofby 2 H NMR on the lipids . Using WALP analogs with this observation is that e¡ects of peptides on phase behaviorTrp at di¡erent positions in the peptide, it was shown that at high P/L can be used as a tool to determine the e¡ectiveinterfacial anchoring interactions can even dominate over hy- length of peptides under negative mismatch conditions (seedrophobic mismatch e¡ects in determining mismatch re- Section 3.3).sponses . In contrast, Lys does not have a speci¢c prefer-ence for the interface  and indeed does not appear to resist 3.2. Backbone conformationdisplacement from the interface towards the aqueous phase In principle, the peptide backbone could adapt to a negativeupon introducing a positive mismatch. Mass spectrometry mismatch by stretching to form a 310 helix. However, as withmeasurements on relatively long KALP peptides suggested positive mismatch, also under a range of negative mismatchthat a signi¢cant part of the hydrophobic transmembrane conditions, a very stable K-helical conformation of backbone(Leu-Ala)n sequence is exposed to the polar region under con- was observed for WALP peptides , further supporting theditions of positive mismatch, suggesting that the energy cost notion that backbone adaptation is an unfavorable mismatchof hydrophobic mismatch, at least for (Leu-Ala)n , is small as response.compared to that of the various mismatch responses . 3.3. Side chain reorientation and interaction of £anking2.5. E¡ects on lipids residues with interfacial region Lipids could adapt to a positive mismatch by changing their Analogous to the situation of positive mismatch, reorienta-e¡ective length, as suggested by the so-called mattress model tion of side chains near the lipid/water interface under nega-. Indeed 2 H NMR experiments indicated a systematic but tive mismatch conditions could result in an increase of thevery small increase in chain order when WALP peptides with e¡ective hydrophobic length of the peptide. The contributionincreasing hydrophobic length were incorporated in diacyl- of Trp and Lys to the e¡ective peptide length could be inves-phosphatidylcholine (PC) bilayers . For KALP peptides, tigated in detail by comparison of the e¡ects of WALP andno signi¢cant stretching of the acyl chains was observed KALP peptides on lipid phase behavior in di¡erent lipid sys-[11,22]. These results are in line with strong interfacial anchor- tems [9,22^24]. For WALP peptides, the e¡ective length wasing interactions of Trp, but not of Lys. Importantly, the in- found to be independent of the type of lipid used , indi-crease in chain ordering observed with the WALP peptides cating that the Trp residues do not change their orientation.was insu⁄cient to compensate for the mismatch, implying However, for KALP peptides large lipid-dependent di¡erencesthat there is also a signi¢cant energy cost to acyl chain in the e¡ective length were observed: in lipids, which by them-stretching. selves have a tendency to form non-lamellar phases, the e¡ec- tive length was much smaller than in PC, which is a typical3. Consequences of negative mismatch for protein and lipid bilayer forming lipid . This suggested that in PC, but not organization in the other lipids, upon inducing a negative mismatch, Lys increases the e¡ective length of the transmembrane segment3.1. Aggregation by snorkeling. This means that the backbone CK is buried in Like with positive mismatch, also under negative mismatch the hydrophobic region of the bilayer and the side chainconditions, when the hydrophobic part of the peptide is small stretches out with its hydrophobic part still interacting withas compared to the bilayer thickness, lysine-£anked peptides the hydrophobic region of the bilayer, but with its positivelyhave a tendency to oligomerize, as shown by £uorescence charged amino group extended outwards into the more polarmeasurements [6,10], and the amount of peptide that can be interfacial region. In systems that by themselves have a ten-stably incorporated in a lipid bilayer decreases with the extent dency to form non-lamellar phases, it was found that theof negative mismatch . For WALP peptides £uorescence system reacts to a mismatch by promoting the formation ofmeasurements  and visualization of striated domains by these non-lamellar phases, rather than by snorkeling. ThisAFM  suggested the formation of linear peptide aggre- behavior is illustrated in Fig. 3. This interpretation was sup-gates under conditions of negative mismatch. ported by experiments with modi¢ed KALP peptides, contain- Like with positive mismatch, at higher P/L and/or at in- ing Lys derivatives with a shortened side chain [23,24]. Fromcreasing mismatch, the tendency to aggregate leads to the these and related experiments, the energetic cost of snorkelingformation of macroscopic aggregates, and peptide-rich aggre- of Lys side chains was estimated and was found to be rathergates can be isolated. Under negative mismatch conditions low, less than 0.7 kcal/mol .these aggregates were found to consist of a well-de¢ned Comparison of the e¡ective lengths of WALP and KALPnon-lamellar phase: the inverted hexagonal (HII ) phase . peptides in PC lipids furthermore suggested that upon de-Formation of such non-lamellar phases can be a way to adapt creasing the peptide length, Trp resists partioning with itsto mismatch. This is explained in a model of the HII phase  indole NH group below the carbonyl region, while the pos-in which the WALP peptides span the distance between ad- itively charged amino group of Lys resists partioning belowjacent tubes, and are surrounded by lipids that, due to the the level of the phosphate .
J.A. Killian/FEBS Letters 555 (2003) 134^138 137 mismatch and hence the strength of the mismatch response. Studies using peptides with other £anking residues suggested that there are subtle di¡erences between the e¡ects of the positively charged residues Lys, Arg and His (at low pH) on one hand, and between the e¡ects of the aromatic Trp, Tyr and Phe on the other hand [23,24]. A number of studies have been carried out to investigate the importance of other param- eters, such as hydrophobicity , but still more systematic studies are required to shed light on how all these di¡erent factors may in£uence mismatch responses. The results discussed here have illustrated how studies on model peptides can provide detailed insight into the variousFig. 3. Schematic illustration of the di¡erences in the e¡ective length mismatch responses. One may wonder how well synthetic pep-of KALP peptides in bilayer lipids (A) and lipids that have a ten- tides represent ‘real’ membrane proteins. The contribution ofdency to form non-lamellar phases (B). Under matching conditions, the transmembrane domains to the various mismatch re-Lys is not snorkeling and the lipids are in a bilayer organization. sponses as observed for the model peptides is likely to beUpon introducing a small negative mismatch, in system A the Lysside chain adapts by snorkeling, thereby increasing the e¡ective similar to responses of single-span membrane proteins. How-length of the peptide, while the lipids remain in a bilayer organiza- ever, synthetic single-span peptides may be less suitable astion. In system B the lipids react by forming a non-lamellar phase, models for multi-span proteins. For example, in multi-spanthereby e¡ectively decreasing their acyl chain length, and the Lys proteins the e¡ects on lipids may be larger and molecularside chain does not snorkel. sorting may occur more easily , or tilting may be a more favorable response, because it may less disturb lipid packing.3.4. E¡ects on lipids On the other hand, the lipid exposed parts will sense mis- 2 H NMR experiments showed that WALP peptides cause a match e¡ects similar to single-span proteins and may causesystematic, but only slight decrease in chain order under con- responses like aggregation and reorientation of side chainsditions of negative mismatch . Since this response is small near the interface, similar to those observed for the modelas compared to what would be required to fully compensate peptides. One indication that anchoring residues behave veryfor a mismatch, this suggests a signi¢cant energy cost to dis- similar in synthetic peptides as in ‘real’ membrane proteinsordering of the lipids, at least when present in a bilayer. For- came from a comparison between model membrane studiesmation of non-lamellar phases, which can occur in PC lipids as discussed in this review and studies on interfacial anchoringat high P/L [8,22], is an e¡ective response to a negative hydro- of proteins upon insertion into microsomal membranes .phobic mismatch and may be regarded as an ultimate lipid Finally, it should be noted that systematic studies on modelchain disordering e¡ect. peptides also can be useful to investigate general principles of peptide/peptide and peptide/lipid interactions. An example is a4. Conclusions and perspectives recent study on the e¡ects of transmembrane segments of proteins on lipid £op, in which WALP and KALP peptides From the results discussed here, it is clear that even in were used as models . Such studies will lead to a bettersingle-lipid systems and using simple synthetic peptides, understanding of the way in which proteins integrate in mem-many di¡erent mismatch responses can be observed, each branes, of how their behavior can be in£uenced by the sur-with its own energy cost. The ¢nal mismatch response, or rounding lipids, and of how proteins can a¡ect lipid organi-combination of responses, will be determined by the balance zation and dynamics.of the energy costs of the individual responses and the energycost of hydrophobic mismatch itself. This will obviously de- Acknowledgements: I thank the many PhD students, post-docs, andpend on many factors. Not only the extent of mismatch will international collaborators who have contributed to the work de-contribute, but also the precise composition of the proteins, scribed here, and I would like to thank Prof. Ben de Kruij¡ for help- ful comments on the manuscript.including their total hydrophobicity, the distribution of sidechains along the helix axis, and the nature of their £ankingresidues, as well as the precise composition of the lipids, in- Referencescluding the size and charge of their head groups and thelength and degree of unsaturation of their acyl chains. In  Lee, A.G. (2003) Biochim. Biophys. Acta 1612, 1^40.  Dumas, F., Lebrun, M.C. and Tocanne, J.F. (1999) FEBS Lett.the studies discussed here, only the role of the £anking resi- 458, 271^277.dues was investigated systematically, by comparison of the  De Planque, M.R.R. and Killian, J.A. (2003) Mol. Membr. Biol.e¡ects of WALP and KALP peptides. 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