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Transmembrane vs. non-transmembrane hydrophobic helix topography in model and natural membranes
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Transmembrane vs. non-transmembrane hydrophobic helix topography in model and natural membranes Erwin London and Khurshida Shahidullah Experimental studies have begun to define how formation of transmembrane (TM) vs. non-TM hydrophobic helix topographies is controlled. It has been found that topography is very sensitive to sequence and lipid structure. Interestingly, there is a broad agreement between studies using artificial model membranes and natural membranes. These studies provide important insights into membrane protein insertion and function. Addresses Department of Biochemistry and Cell Biology, Stony Brook, NY 11794-5215, United States Corresponding author: London, Erwin ([email protected])
Current Opinion in Structural Biology 2009, 19:464–472 This review comes from a themed issue on Engineering and Design Edited by Bill DeGrado and Brian Kuhlman Available online 7th August 2009 0959-440X/$ – see front matter # 2009 Elsevier Ltd. All rights reserved. DOI 10.1016/j.sbi.2009.07.007
Topography and its biological significance The structure and function of the helical hydrophobic segments of membrane proteins are impacted by their interaction with the membrane bilayer. One approach to characterize how a hydrophobic sequence interacts with membranes is to define its topography in terms of the degree to which forms non-TM states (either dissolved in aqueous solution or bound to a membrane surface), or forms a membrane-spanning TM state (Figure 1A). Multiple substates can also exist, including co-existing TM states with different insertion boundaries (i.e. with different transverse positions) and membrane-bound non-TM states that vary in the extent that they penetrate the bilayer, or tilt relative to the surface. Much recent progress has been made characterizing these topographies with synthetic hydrophobic sequences and model membranes [1–3,4,5–10]. However, in nature membrane insertion usually occurs within the translocon complex. Recently, methods allowing the analysis of the sequence dependence of translocon-dependent TM insertion have been developed [11,12]. Topography studies should help answer fundamental questions concerning membrane protein insertion. How do sequence and lipid composition control topography? Current Opinion in Structural Biology 2009, 19:464–472
To what extent does insertion in artificial and natural membranes differ? Is the structure of a hydrophobic helix in natural membranes kinetically controlled by the translocon, or does it reflect the energetics of its interactions with lipid bilayers? With regard to these points it is important to note that even in a lipid bilayer equilibration between TM and non-TM states cannot always occur. When a hydrophobic sequence is bounded on both sides by large hydrophilic domains, equilibration requires membrane translocation of a large hydrophilic domain. This self-translocation is only possible for specialized proteins, for example, certain protein toxins, although it may occur under some other conditions for ordinary proteins [13]. TM/non-TM interconversion is more facile for isolated hydrophobic sequences bounded on one side by only a few hydrophilic residues, or hydrophilic helical hairpins with a short hydrophilic connecting loop (Figure 1B). Studies of the sequence and lipid dependence of topography should also provide insights into post-translational topography changes. These are known to occur in a number of cases. The helices of diphtheria toxin T domain convert from a membrane-bound non-TM state to a TM hairpin thought to be essential for membrane translocation of its A chain [14,15]. Similar behavior has been observed for colicins and proposed for Bcl family proteins [16]. TM/non-TM interconversion in these cases is generally coupled to large-scale conformational changes in other parts of the protein [17]. Post-translational TM insertion has also been reported for proteins with a helixtype hydrophobic anchoring (tail) sequences capped by a short hydrophilic sequence [18,19]. The switch of helixforming antimicrobial peptides from membrane-bound non-TM states to TM states can be crucial for their pore formation [20]. In other cases the prevention of the stable TM insertion of a hydrophobic sequence may be crucial for function [21,22]. There may be many as yet unidentified cases of TM/nonTM interconversion. Genomic analysis suggests considerable overlap in hydrophobicity between TM and soluble sequences [23,24]. However, the behavior of sequences in the translocon suggests low overlap in hydrophobicity between secreted sequences and TM helices [12]. Even so, where the energies of TM and non-TM states are similar, significant amounts of both TM and non-TM states with distinct conformations and functions may form, as in the case of the PrP (prion) protein, where conformationally distinct TM and non-TM states may have important consequences for pathogenesis [25]. www.sciencedirect.com
Transmembrane vs. non-transmembrane hydrophobic helix topography London and Shahidullah 465
Figure 1
TM/non-TM interconversion. Hydrophobic sequences are represented by gray rectangles. A. Equilibria between different states. Equilibria between different secondary structures can also occur, but are not illustrated. B. TM/non-TM equilibration can be blocked by large hydrophilic domains that cannot cross membranes (top), but not by very short hydrophilic sequences at the end of a hydrophobic helix (middle) or middle of a hydrophobic hairpin (bottom). C. TM helix position can be affected by anchoring. When anchoring is weak the distance of the residue in the center of a hydrophobic sequence (here Trp (W)) TM will be shallower than when anchoring is strong (right).
Finally, studies of topography can lead to the design of hydrophobic helices with biomedical applications. The pHLIP peptide, which converts from a soluble to TM state at low pH, is a tumor marker, inserting specifically into the membrane of tumor cells because of their acidic milieu [26].
Distinguishing transmembrane from nontransmembrane topography Hydrophobic helix topography in model membranes can be studied by NMR [1,8,27], neutron diffraction [28–30], attenuated total reflection Fourier transform infrared spectroscopy [31–33] and oriented circular dichroism [34–36]. The latter two methods do not require isotopic substitution, and have high sensitivity. Differential scanning calorimetry can also evaluate topography, as only the TM state strongly perturbs the gel to liquid disordered melting transition of a lipid bilayer [6,37,38]. This method should probe the average of peptide behavior above and below the melting transition. Fluorescence spectroscopy is another sensitive method widely used for topography studies [7,9,10]. The membrane location of a Trp (as gauged by lmax and/or fluorwww.sciencedirect.com
escence quenching) when placed at the center of a hydrophobic helix can distinguish TM topography (Trp deep) from non-TM topography (Trp shallow). Combining lmax and quenching distinguishes cases in which a Trp has an intermediate depth from that in which non-TM and TM populations co-exist [2]. (NMR also readily detects co-existing TM and non-TM populations [1].) One disadvantage of fluorescence is that Trp may perturb the behavior of a sequence. This can be largely subtracted out when the differences between two Trp-bearing helices are studied. Correcting for alteration of hydrophobicity by Trp, or demonstrating that a Trp does not alter function can also rule out perturbation effects [2,19]. In natural membranes translocon-controlled topography can be studied [11,12,38,39,40,41]. This allows rapid investigation of a wide variety of sequences in a biological milieu. In one approach, protection from proteolysis is used to identify TM topography [40]. In another approach a test hydrophobic sequence flanked by N-linked glycosylation sites is introduced into a chimeric protein and the topography of newly synthesized protein is quantified by the levels of single (TM), and double (non-TM) Current Opinion in Structural Biology 2009, 19:464–472
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glycosylation. This is evaluated on SDS-PAGE via differences in molecular weight and quantitative staining. Studies in natural and model membranes have different strengths. Translocon-induced insertion is likely to define topographies that are relevant in vivo, while lipid composition and pH can be varied and their effects studied most easily using model membranes. However, there has been recent progress in controlling natural membrane lipid composition [13]. The properties of non-TM states are also most easily defined in model membranes. Finally, model membranes may be useful for understanding changes in topography occurring after the release of a membrane protein from the translocon. To measure the sequence dependence of topography TM and non-TM states must co-exist in measurable amounts. This can be achieved by controlling the hydrophobicity of a host sequence into which residues of interest are substituted [11]. Another method is to vary pH using hydrophobic sequences containing an ionizable residue, which destabilizes the TM state when charged [32]. A third method is to vary bilayer width [2]. The TM state is destabilized when bilayer width exceeds hydrophobic helix length (i.e. conditions of negative mismatch).
TM hydrophobicity threshold in allhydrophobic helices Studies of translocon-induced membrane insertion and of topography in zwitterionic phosphatidylcholine (PC) model membranes show that polyAla sequences are generally not hydrophobic enough to form a stable TM state [6,42,43]. In translocon experiments, in one study 19 Ala long sequences did form predominantly (>50%) TM structure [40]. However, the SerPhe sequence following the Ala might have slightly aided the formation of a TM topography. In another study, the presence of 3–5 Leu in a 19 residue long hydrophobic sequence otherwise composed of Ala was necessary for 50% TM topography (i.e. equal levels of TM and non-TM states) [11]. An analysis of genomic data showed that all-Ala hydrophobic sequences cannot form a stable TM structure, and 1–2 Leu in such sequences are required to form a TM state [23]. (This analysis included multi-TM helix proteins, in which helix–helix interaction can stabilize the TM state (see below).) In PC model membranes, it was found that 7 Leu mixed with 15 Ala (with Leu all throughout the hydrophilic sequence, but also all on one face of the helix) were barely enough for TM insertion [37]. Studies of Lys-flanked hydrophobic sequences showed that a polyAla sequence with or without a few Leu forms a stable TM state in vesicles containing anionic lipids, but does not in vesicles composed solely of PC [44]. In another study, no Current Opinion in Structural Biology 2009, 19:464–472
significant difference between TM stability was found for an alternating LeuAla sequence and an equivalent composition with a segment of Leu followed by a segment of Ala [45]. However, an amphiphilic arrangement of Ala and Leu might help stabilize a surface-bound nonTM state relative to a TM state, similar to its effect on the surface state stability relative to an unbound state [46].
Effect of hydrophilic residues in hydrophobic helices Using the mammalian translocon system and a sequence in which each type of amino acid was placed in the middle of a 19 residue LeunAlam sequence, a complete ‘biological hydrophobicity’ scale was derived [11]. From most to least hydrophobic, an order of I,L > F,V > C,M > A > W > T > Y > G > S > N,H > P > Q > R > E,K > D was determined. A second study showed that the scale was not greatly affected by whether the N or C-terminus of the LeunAlam sequence faced the cytosol [47]. A similar (but not identical) order was also later found in the yeast translocon and YidC-dependent insertion in E. coli [38,39]. Biological hydrophobicity values were well correlated (r = 0.85) with those from octanol–water partitioning [11], a direct measure of amino acid residue hydrophobicity. This implies that hydrophobicity is the primary factor determining participation of residues in TM helices. Furthermore, biological hydrophobicity values parallel the relative abundance of residues that had been found in an early study of TM segments in single TM helix proteins [48]. This relationship is reinforced by the similarity (r = 0.96) between biological hydrophobicity and the TM tendency scale [23]. TM tendency is a measure of the tendency of a residue to favor TM insertion derived from the compositions of sequences in databases of soluble and TM segments, using a novel analysis yielding a near-theoretical limit of accuracy. Thus, biological hydrophobicity reflects properties that apply to more than just the simple sequences. Model membrane measurements of the effects of hydrophilic residues introduced into hydrophobic helices is less complete [1,5,49]. TM insertion in polyAla sequences containing three ‘guest’ residues was found to decrease in the order I > L > G > S [9]. This could reflect both altered membrane binding (K1 in Figure 1) and alteration of the TM/surface topography equilibrium (K2 in Figure 1). In a study using fully membrane-associated Leu-rich hydrophobic peptides containing two substitutions, TM topography decreased in the order L > G > S > P > K > D [2]. With single substitutions the TM state was more stable, but it could still be discerned that His, Pro, Asp and Lys destabilized TM topography more than Ala, Gly, Phe or Ser. Notice that the order of destabilization of the TM state in these studies mirrors that in the translocon-based studies. In www.sciencedirect.com
Transmembrane vs. non-transmembrane hydrophobic helix topography London and Shahidullah 467
a third study, four substitutions were introduced into peptides containing a mixture of Leu and Ala [32]. Most of the substitutions tested were relatively hydrophobic. It was found that Trp, and especially Tyr, destabilized the TM state, relative to a surface-bound state, much more than other hydrophobic residues, consistent with their known preference for the polar–non polar interface of the bilayer. Other results (e.g. weaker stabilization of the TM state by Val than by Ser or Ala) were surprising, and might have been affected by steric constraints when four substitutions are placed on the same face of a helix [32].
Effect of ionizable (‘charged’) residues in hydrophobic helices When charged, ionizable residues (Asp, Glu, Lys, Arg, His) in hydrophobic helices should destabilize the TM state most strongly. Model membrane studies showed a single Asp residue near the center of a hydrophobic sequence containing mostly Leu prevents formation of TM topography when Asp is charged, but not when it is uncharged [5,49]. Near pH 7, a single Lys in highly hydrophobic sequences did not prevent the formation of a TM topography, while with two Lys, TM topography was of borderline stability, and with more than two the TM state did not form. Unfortunately, it is not clear if the Lys was charged [2,8]. Another study showed four charged His are sufficient to destroy TM orientation in an alternating LeuAla hydrophobic sequence, but four uncharged His are not [1]. How much do uncharged ionizable residues destabilize TM topography? A clue comes from a study mainly designed to measure the relative tendency of hydrophilic residues to shift TM helix transverse position [4]. The effects of negative mismatch upon the stability of TM topography indicated that both uncharged Asp and uncharged His destabilized TM topography similarly to Asn or Gln, while uncharged Glu was more similar to Ser [4].
Effect of hydrophilic residue sequence position How the position of a specific residue within a hydrophobic sequence alters TM insertion was systematically analyzed in the translocon system [12,11]. A smooth dependence of the extent of TM helix topography vs. residue position was found, with hydrophilic residues destabilizing TM helix topography most strongly when at the center of a hydrophobic sequence (i.e. when located at the bilayer center). The position dependence was strong for the most highly hydrophilic residues, while a somewhat flatter, inverted position dependence was found for hydrophobic residues. As expected, Trp and Tyr promoted TM helix topography most when close to the edge of the hydrophobic sequence. Overall, the profiles were very similar to those defined by statistical analysis of the abundance of different residues vs. diswww.sciencedirect.com
tance from the center of a hydrophobic segment for membrane proteins of known structure [12,50,51]. It must be cautioned that a single hydrophilic residue in a TM helix can cause it to undergo a transverse shift in location in order to locate the hydrophilic residue close to the bilayer surface [4,52–55]. These studies showed that in the TM state the relationship between the position of a hydrophilic residue in a hydrophobic sequence and its membrane depth is not trivial, and depends on hydrophilic residue type. In other words, equivalent positions of different hydrophilic residues in a sequence do not mean they have equivalent membrane depths. Transverse shifts should tend to mask what would otherwise be larger changes in TM/non-TM equilibrium. One translocon study examined the effect of pairs of guest hydrophilic residues, symmetrically varying their position relative to the center of the hydrophobic sequence [11]. This should tend to lessen transverse shifts, which would move one hydrophilic residue toward the membrane surface but pull the other hydrophilic residue toward the bilayer center, largely canceling out any energetic advantage of transverse shift.
TM helix minimum length It is generally assumed that TM proteins require nearly 20 residues to span the membrane hydrocarbon core. However, TM helices with shorter hydrophobic segments form, and their behavior is of interest because of their potential role in length-dependent sorting of TM sequences between Golgi (which averages 15 residue TM sequences) and plasma membrane (18–20 residues) [10,56,57]. In both bacterial and eukaryotic membranes sequences with as few as 10 or less consecutive hydrophobic residues form TM segments [40,58,59]. A glycosylation study found that a C-terminal tail of 12 Leu residues was sufficient to act as a TM anchor for synaptobrevin2 [60]. Several model membrane studies demonstrated that strong negative mismatch prevented TM insertion of highly hydrophobic peptides [2,10,27,49]. Model membrane studies using protocols in which the TM and membrane-bound non-TM states were in equilibrium also found TM topography was destabilized by negative mismatch, with 11 highly hydrophobic residues having borderline TM stability in bilayers with a physiologically relevant width, that is, composed of dioleoyl PC (DOPC). A value of 11–12 residues was also found for the minimum continuous hydrophobic segment needed to form TM topography when charged residues interrupted long hydrophobic sequences [2,49]. Another study showed that 10 hydrophobic residues were too short to form a TM helix in DOPC bilayers [27]. A detailed recent study in model membrane vesicles found that in DOPC 50%TM topography required a Current Opinion in Structural Biology 2009, 19:464–472
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minimum length of 11–12 residues for Leu sequences and 13 residues for less hydrophobic alternating Leu–Ala sequences [45,61]. As expected, longer lengths were necessary to form TM states in thicker bilayers. Charged flanking residues increased the minimum TM-forming length relative to uncharged hydrophilic residues. On the basis of energetic considerations it was predicted that minimum TM-forming length would increase greatly and be more hydrophobicity-dependent for sequences even more weakly hydrophobic than alternating Leu–Ala. Similar conclusions were obtained in a translocon study [12]. For 50% TM topography, the minimum length was near 10 for all-Leu hydrophobic sequences, 13 for sequences that were nearly half Leu and half Ala, and much more for less hydrophobic sequences, rising to 25 or more for hydrophobic sequences composed only of Ala. Somewhat smaller numbers for each of these cases were obtained in an earlier study, in which the hydrophobic sequences contained an additional SerPhe at the C-terminal, but the overall pattern was the same [40]. The effect of negative mismatch was found to have similar affects on the stability of the TM state for hydrophobic antibiotic peptides of the ‘peptiabol’ class [62]. These molecules are rich in the unusual hydrophobic amino acid a-aminoisobutyric acid. The 15-residue peptaibol ampullosporin A only formed a TM helix in very thin bilayers, while the 20-residue peptaibol alamethicin formed a TM state in bilayers with a medium width. How are short TM helices accommodated in a membrane? Adjustment of local bilayer width is certainly one important mechanism [63–65]. Partial burial of flanking residues in the membrane and helix oligomerization are also likely to be important [63].
Effect of residues flanking the TM sequence upon topography Residues surrounding (‘flanking’) the ends of a hydrophobic sequence can also affect topography. If these residues are much more hydrophilic than those within the sequence, they are considered to be outside of the hydrophobic sequence. However, if they are modestly hydrophilic the definition of the ends of a TM sequence can be arbitrary. One can define the overall sequence as having a short, more highly hydrophobic segment or a longer and less hydrophobic segment. In any case, model membrane experiments described above showed that increased hydrophilicity of the residues at the ends of a short hydrophobic sequence destabilized TM topography [45]. In the translocon system stabilization of TM topography was observed for hydrophobic sequences having cationic sequences at their C-terminal flanking side, which locates in the cytofacial leaflet [66]. These effects were observed even when the cationic residues and hydrophobic helix were separated by 10–15 apparently disordered Gly and Pro rich sequences. This stabilCurrent Opinion in Structural Biology 2009, 19:464–472
ization of the TM state may partly reflect favorable electrostatic interactions with anionic lipids on the cytofacial leaflet (see below). Destabilization of TM topography was observed with anionic residues on an N-terminal flanking side of a hydrophobic helix, which locates in the exofacial leaflet.
Effect of helix–helix interaction upon topography Interactions between TM, and non-TM, helices should also affect the TM/non-TM equilibrium. Both Asn–Asn and Asp–Asp interactions have been studied in the translocon system, and a significant stabilization of the TM topography was observed [67]. No doubt such helix-helix interactions are a major factor stabilizing TM topography for helices of multi-TM helix proteins, in which hydrophilic residues are often present for functional reasons.
Lipid composition dependence of topography The effect of lipid composition upon hydrophobic sequence binding to model membranes (i.e. K1, Figure 1A) has been much studied. Anionic lipids strongly promote membrane binding of hydrophobic peptides and proteins containing cationic residues [9]. The effect of lipid composition upon the equilibrium between membrane-bound TM and non-TM states (i.e. K2, Figure 1A) has only been studied recently [44]. The presence of anionic lipids in model membranes (at as low as 10 mol%, within the physiological range) was found to strongly stabilize TM topography for hydrophobic helices flanked by cationic residues. That this effect was electrostatic (i.e. Coulombic) was confirmed using His-flanked peptides. Stabilization of the TM configuration by anionic lipid was only observed at pH values at which His was charged. A surprising difference between behavior in model membranes containing phosphatidylserine (PS) and phosphatidylglycerol (PG) was observed [44]. Although both lipids are anionic, the depth of a Trp at the center of a hydrophobic sequence was much deeper in PS-containing vesicles than in PG-containing vesicles. Either PS stabilized the TM configuration to a greater degree than PG, or PS anchored the TM state such that the Trp was in a more fixed position at the bilayer center (Figure 1C). Interestingly, circular dichroism showed significantly higher helix content in PS-containing vesicles than in those containing PG or PC. This may offer clues into differences in the function of different anionic lipids. The effect of different zwitterionic (i.e. PC or phosphatidylethanolamine) or uncharged lipids (i.e. cholesterol) on the equilibrium between the membrane-bound nonTM and TM states was small, at most [44]. However, in another study using lipids with long monounsaturated acyl chains, cholesterol was found to destabilize TM www.sciencedirect.com
Transmembrane vs. non-transmembrane hydrophobic helix topography London and Shahidullah 469
topography [68]. This could be due to acyl chain straightening by cholesterol increasing negative mismatch [68]. Tighter lipid packing in the presence of cholesterol may also be involved. Methods to prepare vesicles with asymmetric lipid compositions in their inner and outer leaflets were recently developed [69]. It was found that an asymmetric distribution of anionic lipids promoted the formation of TM topography by a cationic residue-flanked hydrophobic helix [69]. Asymmetric model membranes that mimic cell membranes may be useful for more precise studies of how lipid bilayer physical state controls TM helix behavior (and function), including studies of the effects of bilayer width and lipid packing in liquid ordered membrane domains (lipid rafts).
Issues complicating comparison of the stabilities of different topographies in translocon and model membrane-based experiments Several factors complicate comparison of model membrane and translocon experiments. The sequences studied in the translocon and model membrane systems have been similar, but not identical. Another difference is that isolated helices are used in model membranes and chimeric proteins in the translocon. Furthermore, translocon and model membrane studies have used different hydrophilic flanking residues. Comparison of translocon-induced and model membrane insertion is also complicated by differences in terms of secondary structures of the non-TM state, and whether it is exposed to aqueous solution, buried within a protein, or bound to the membrane surface. The difference between the TM and non-TM energies should be largest when the non-TM state is fully exposed to aqueous solution. This might help explain the smaller difference in the TM/nonTM dependence on hydrophobicity measured in yeast relative to that in mammalian membranes [11,39]. Further complicating interpretation differences in TM insertion, the stability of a surface-bound state can be affected by which residues point toward solution and which point toward the center of the lipid bilayer, and how deeply the surface-bound state is buried in the bilayer. Crowding on the membrane surface due to unfavorable peptide– peptide interactions [70] or strain arising from peptideinduced expansion of the leaflet into which insertion occurs could destabilize surface-bound non-TM states. None of these factors would affect a non-TM state in solution. Another crucial issue is whether experimental topography reflects an equilibrium. In most model membrane experiments, the peptides used are water insoluble, and to avoid irreversible aggregation in solution samples are prepared by mixing peptide and lipid before vesicle www.sciencedirect.com
formation. Demonstration that the TM state is in facile equilibrium with a membrane-bound non-TM state has been shown by reversibly altering the TM/non-TM balance by altering bilayer width (via binding and dissociation of decane [68,71] or pH [5,32,49]), using highly hydrophobic sequences with only a couple of charged flanking residues. However, the behavior of water-soluble Lysflanked Ala-rich peptides suggest they face a kinetic barrier to TM insertion when added to preformed membranes [72]. Further studies of what sequences allow or block posttranslational TM/non-TM equilibration is a high priority. This knowledge could be used in concert with mutagenesis to control TM/non-TM interconversion in intact proteins, and so investigate its function. Whether translocon-induced insertion represents an equilibrium is unclear, although its correlation with both hydrophobicity and model membrane behavior suggests this might be the case.
Why do translocon and model membrane systems give similar results? Given the issues above, the similarity between the insertion in natural and model membranes is remarkable. It may be due to interaction of potential TM segments with the lipid bilayer during their residence in the translocon [11]. It is also possible that the properties of the walls of the translocon ‘pore’ (which are depleted in ionizable residues, but not polar residues) mimic the lipid bilayer to a limited degree.
Acknowledgement This work was supported by NIH grant GM 48596.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1.
Bechinger B: Towards membrane protein design: pH-sensitive topology of histidine-containing polypeptides. J Mol Biol 1996, 263:768-775.
2.
Caputo GA, London E: Cumulative effects of amino acid substitutions and hydrophobic mismatch upon the transmembrane stability and conformation of hydrophobic alpha-helices. Biochemistry 2003, 42:3275-3285.
3.
de Planque MR, Killian JA: Protein-lipid interactions studied with designed transmembrane peptides: role of hydrophobic matching and interfacial anchoring. Mol Membr Biol 2003, 20:271-284.
4.
Krishnakumar SS, London E: The control of transmembrane helix transverse position in membranes by hydrophilic residues. J Mol Biol 2007, 374:1251-1269. Hydrophilic residues within TM helices can induce a transverse shift so that the hydrophilic residue locates closer to the membrane surface while TM structure is maintained. In this fluorescence study hydrophilic residues were ranked in terms of the magnitude of shifts they induced in model membranes. The stability of the TM state under conditions of negative mismatch was found to decrease in a fashion related to the degree to which the transverse shifts shortened the length of the membrane-spanning sequence. Current Opinion in Structural Biology 2009, 19:464–472
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5.
Lew S, Ren J, London E: The effects of polar and/or ionizable residues in the core and flanking regions of hydrophobic helices on transmembrane conformation and oligomerization. Biochemistry 2000, 39:9632-9640.
6.
Lewis RN, Zhang YP, Hodges RS, Subczynski WK, Kusumi A, Flach CR, Mendelsohn R, McElhaney RN: A polyalanine-based peptide cannot form a stable transmembrane alpha-helix in fully hydrated phospholipid bilayers. Biochemistry 2001, 40:12103-12111.
7.
Ren J, Lew S, Wang Z, London E: Transmembrane orientation of hydrophobic alpha-helices is regulated both by the relationship of helix length to bilayer thickness and by the cholesterol concentration. Biochemistry 1997, 36:10213-10220.
8.
Vogt B, Ducarme P, Schinzel S, Brasseur R, Bechinger B: The topology of lysine-containing amphipathic peptides in bilayers by circular dichroism, solid-state NMR, and molecular modeling. Biophys J 2000, 79:2644-2656.
9.
Liu LP, Deber CM: Anionic phospholipids modulate peptide insertion into membranes. Biochemistry 1997, 36:5476-5482.
10. Webb RJ, East JM, Sharma RP, Lee AG: Hydrophobic mismatch and the incorporation of peptides into lipid bilayers: a possible mechanism for retention in the Golgi. Biochemistry 1998, 37:673-679. 11. Hessa T, Kim H, Bihlmaier K, Lundin C, Boekel J, Andersson H, Nilsson I, White SH, von Heijne G: Recognition of transmembrane helices by the endoplasmic reticulum translocon. Nature 2005, 433:377-381. 12. Hessa T, Meindl-Beinker NM, Bernsel A, Kim H, Sato Y, Lerch Bader M, Nilsson I, White SH, von Heijne G: Molecular code for transmembrane-helix recognition by the Sec61 translocon. Nature 2007, 450:1026-1030. Using chimeric proteins and glycosylation-based topography determination this report defined the affect of every amino acid on the amount of TM topography formation in mammalian membranes as a function of the position of the residue within a hydrophobic sequence. In addition, the effect of the length of sequences composed of Leu and Ala necessary to form a TM state was measured as a function of hydrophobicity, which was controlled by the relative content of these two amino acids.
diacylglycerol kinase epsilon in a cell membrane and its relationship to predictions from modeling. J Mol Biol 2008, 383:797-809. 22. Dalley JA, Bulleid NJ: The endoplasmic reticulum (ER) translocon can differentiate between hydrophobic sequences allowing signals for glycosylphosphatidylinositol anchor addition to be fully translocated into the ER lumen. J Biol Chem 2003, 278:51749-51757. 23. Zhao G, London E: An amino acid ‘transmembrane tendency’ scale that approaches the theoretical limit to accuracy for prediction of transmembrane helices: relationship to biological hydrophobicity. Protein Sci 2006, 15:1987-2001. 24. Cunningham F, Rath A, Johnson RM, Deber CM: Distinctions between hydrophobic helices in globular proteins and transmembrane segments as factors in protein sorting. J Biol Chem 2009, 284:5395-5402. 25. Hegde RS, Mastrianni JA, Scott MR, DeFea KA, Tremblay P, Torchia M, DeArmond SJ, Prusiner SB, Lingappa VR: A transmembrane form of the prion protein in neurodegenerative disease. Science 1998, 279:827-834. 26. Andreev OA, Dupuy AD, Segala M, Sandugu S, Serra DA, Chichester CO, Engelman DM, Reshetnyak YK: Mechanism and uses of a membrane peptide that targets tumors and other acidic tissues in vivo. Proc Natl Acad Sci USA 2007, 104:7893-7898. The artificial pHLIP peptide, derived from a TM sequence found in bacteriorhodopsin, forms a TM state only at low pH owing to the presence of Asp residues in its hydrophobic sequence. It was found to accumulate several-fold in mouse tumors, and owing to the low pH in the tumor tissue. Peptides with Lys or Asn in place of Asp, did not have this property. 27. Harzer U, Bechinger B: Alignment of lysine-anchored membrane peptides under conditions of hydrophobic mismatch: a CD, 15N and 31P solid-state NMR spectroscopy investigation. Biochemistry 2000, 39:13106-13114. 28. Han X, Mihailescu M, Hristova K: Neutron diffraction studies of fluid bilayers with transmembrane proteins: structural consequences of the achondroplasia mutation. Biophys J 2006, 91:3736-3747.
13. Bogdanov M, Xie J, Dowhan W: Lipid-protein interactions drive membrane protein topogenesis in accordance with the positive-inside rule. J Biol Chem 2008, 284:9637-9641.
29. Bradshaw JP, Darkes MJ, Harroun TA, Katsaras J, Epand RM: Oblique membrane insertion of viral fusion peptide probed by neutron diffraction. Biochemistry 2000, 39:6581-6585.
14. Kachel K, Ren J, Collier RJ, London E: Identifying transmembrane states and defining the membrane insertion boundaries of hydrophobic helices in membrane-inserted diphtheria toxin T domain. J Biol Chem 1998, 273:22950-22956.
30. Dante S, Hauss T, Dencher NA: Beta-amyloid 25 to 35 is intercalated in anionic and zwitterionic lipid membranes to different extents. Biophys J 2002, 83:2610-2616.
15. Wang Y, Malenbaum SE, Kachel K, Zhan H, Collier RJ, London E: Identification of shallow and deep membrane-penetrating forms of diphtheria toxin T domain that are regulated by protein concentration and bilayer width. J Biol Chem 1997, 272:25091-25098. 16. Antignani A, Youle RJ: How do Bax and Bak lead to permeabilization of the outer mitochondrial membrane? Curr Opin Cell Biol 2006, 18:685-689. 17. Wang J, Rosconi MP, London E: Topography of the hydrophilic helices of membrane-inserted diphtheria toxin T domain: TH1TH3 as a hydrophilic tether. Biochemistry 2006, 45:8124-8134. 18. Wattenberg B, Lithgow T: Targeting of C-terminal (tail)anchored proteins: understanding how cytoplasmic activities are anchored to intracellular membranes. Traffic 2001, 2:66-71. 19. Fujita K, Krishnakumar SS, Franco D, Paul AV, London E, Wimmer E: Membrane topography of the hydrophobic anchor sequence of poliovirus 3A and 3AB proteins and the functional effect of 3A/3AB membrane association upon RNA replication. Biochemistry 2007, 46:5185-5199. 20. Huang HW: Molecular mechanism of antimicrobial peptides: the origin of cooperativity. Biochim Biophys Acta 2006, 1758:1292-1302. 21. Decaffmeyer M, Shulga YV, Dicu AO, Thomas A, Truant R, Topham MK, Brasseur R, Epand RM: Determination of the topology of the hydrophobic segment of mammalian Current Opinion in Structural Biology 2009, 19:464–472
31. Bechinger B, Ruysschaert JM, Goormaghtigh E: Membrane helix orientation from linear dichroism of infrared attenuated total reflection spectra. Biophys J 1999, 76:552-563. 32. Aisenbrey C, Goormaghtigh E, Ruysschaert JM, Bechinger B: Translocation of amino acyl residues from the membrane interface to the hydrophobic core: thermodynamic model and experimental analysis using ATR-FTIR spectroscopy. Mol Membr Biol 2006, 23:363-374. 33. Aisenbrey C, Kinder R, Goormaghtigh E, Ruysschaert JM, Bechinger B: Interactions involved in the realignment of membrane-associated helices. An investigation using oriented solid-state NMR and attenuated total reflection Fourier transform infrared spectroscopies. J Biol Chem 2006, 281:7708-7716. 34. Wu Y, Huang HW, Olah GA: Method of oriented circular dichroism. Biophys J 1990, 57:797-806. 35. Mykhailiuk PK, Afonin S, Palamarchuk GV, Shishkin OV, Ulrich AS, Komarov IV: Synthesis of trifluoromethyl-substituted proline analogues as 19F NMR labels for peptides in the polyproline II conformation. Angew Chem Int Ed Engl 2008, 47:5765-5767. 36. Burck J, Roth S, Wadhwani P, Afonin S, Kanithasen N, Strandberg E, Ulrich AS: Conformation and membrane orientation of amphiphilic helical peptides by oriented circular dichroism. Biophys J 2008, 95:3872-3881. 37. Lewis RN, Liu F, Krivanek R, Rybar P, Hianik T, Flach CR, Mendelsohn R, Chen Y, Mant CT, Hodges RS et al.: Studies of the www.sciencedirect.com
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minimum hydrophobicity of alpha-helical peptides required to maintain a stable transmembrane association with phospholipid bilayer membranes. Biochemistry 2007, 46:1042-1054. 38. Xie K, Hessa T, Seppala S, Rapp M, von Heijne G, Dalbey RE: Features of transmembrane segments that promote the lateral release from the translocase into the lipid phase. Biochemistry 2007, 46:15153-15161. In this report, proteolysis was used to detect the effect of sequence upon TM insertion induced by the YidC ‘translocon’ of bacteria, which handles different proteins and is distinct from the SecYEG translocon. The latter is analogous to that in mammals. The sequence dependence of YidCdependent insertion was strikingly similar to that induced by the mammalian translocon.
This study used a statistical analysis of membrane proteins with known structures to derive an apparent energy vs. position of a residue introduced into a hydrophobic sequence, similar to that carried out in references [12,50]. When these ‘energies’ were used to predict the behavior of Lys flanking polyLeu sequences, behaviors similar to those previously measured in model membranes were found. 52. Monne M, Nilsson I, Johansson M, Elmhed N, von Heijne G: Positively and negatively charged residues have different effects on the position in the membrane of a model transmembrane helix. J Mol Biol 1998, 284:1177-1183. 53. Nilsson I, Saaf A, Whitley P, Gafvelin G, Waller C, von Heijne G: Proline-induced disruption of a transmembrane alpha-helix in its natural environment. J Mol Biol 1998, 284:1165-1175.
39. Hessa T, Reithinger JH, von Heijne G, Kim H: Analysis of the transmembrane helix integration in the endoplasmic reticulum in S. cerevisiae. J Mol Biol 2009, 386:1222-1228. In this report, glycosylation was used to detect the effect of sequence upon TM insertion induced by the yeast translocon. The affect of different residues upon TM insertion was similar to that induced by the mammalian translocon, but there were notable differences in detail. Most striking was a scaled-down dependence of topography upon hydrophobicity relative to that in mammals.
54. Braun P, von Heijne G: The aromatic residues Trp and Phe have different effects on the positioning of a transmembrane helix in the microsomal membrane. Biochemistry 1999, 38:9778-9782.
40. Kuroiwa T, Sakaguchi M, Mihara K, Omura T: Systematic analysis of stop-transfer sequence for microsomal membrane. J Biol Chem 1991, 266:9251-9255.
56. Bretscher MS, Munro S: Cholesterol and the Golgi apparatus. Science 1993, 261:1280-1281.
41. Hessa T, White SH, von Heijne G: Membrane insertion of a potassium-channel voltage sensor. Science 2005, 307:1427. 42. Bechinger B: Membrane insertion and orientation of polyalanine peptides: a (15)N solid-state NMR spectroscopy investigation. Biophys J 2001, 81:2251-2256. 43. Nilsson I, Johnson AE, von Heijne G: How hydrophobic is alanine? J Biol Chem 2003, 278:29389-29393.
55. Stefansson A, Armulik A, Nilsson I, von Heijne G, Johansson S: Determination of N- and C-terminal borders of the transmembrane domain of integrin subunits. J Biol Chem 2004, 279:21200-21205.
57. Munro S: An investigation of the role of transmembrane domains in Golgi protein retention. EMBO J 1995, 14:4695-4704. 58. Adams GA, Rose JK: Structural requirements of a membranespanning domain for protein anchoring and cell surface transport. Cell 1985, 41:1007-1015. 59. Chen H, Kendall DA: Artificial transmembrane segments. Requirements for stop transfer and polypeptide orientation. J Biol Chem 1995, 270:14115-14122.
44. Shahidullah K, London E: Effect of lipid composition on the topography of membrane-associated hydrophobic helices: stabilization of transmembrane topography by anionic lipids. J Mol Biol 2008, 379:704-718. This fluorescence study examined the effect of lipid composition upon the balance between TM and non-TM topographies in model membranes. Anionic lipids were found to stabilize TM topography for a wide variety of hydrophobic sequences with cationic flanking residues.
60. Whitley P, Grahn E, Kutay U, Rapoport TA, von Heijne G: A 12-residue-long polyleucine tail is sufficient to anchor synaptobrevin to the endoplasmic reticulum membrane. J Biol Chem 1996, 271:7583-7586.
45. Krishnakumar SS, London E: Effect of sequence hydrophobicity and bilayer width upon the minimum length required for the formation of transmembrane helices in membranes. J Mol Biol 2007, 374:671-687.
62. Salnikov ES, Friedrich H, Li X, Bertani P, Reismann S, Hertweck C, O’Neil JD, Raap J, Bechinger B: Structure and alignment of the membrane-associated peptaibols ampullosporin A and alamethicin by oriented 15N and 31P solid-state NMR spectroscopy. Biophys J 2009, 96:86-100. Using NMR methods, this study showed that the effect of negative mismatch upon the stability of the TM state apply to natural bacterial peptides containing aminoisobutyric acid. Unlike alamethicin, the short peptide ampullosporin A only had the ability to form a TM state in very thin bilayers. Additional experiments suggested that these molecules formed mixed 310/a-helices. The authors hypothesize that balance between TM and non-TM states could be closely related to the way that these peptides exert their antibiotic activities.
46. Fernandez-Vidal M, Jayasinghe S, Ladokhin AS, White SH: Folding amphipathic helices into membranes: amphiphilicity trumps hydrophobicity. J Mol Biol 2007, 370:459-470. 47. Lundin C, Kim H, Nilsson I, White SH, von Heijne G: Molecular code for protein insertion in the endoplasmic reticulum membrane is similar for N(in)-C(out) and N(out)-C(in) transmembrane helices. Proc Natl Acad Sci USA 2008, 105:15702-15707. 48. Landolt-Marticorena C, Williams KA, Deber CM, Reithmeier RA: Non-random distribution of amino acids in the transmembrane segments of human type I single span membrane proteins. J Mol Biol 1993, 229:602-608. 49. Caputo GA, London E: Position and ionization state of Asp in the core of membrane-inserted alpha helices control both the equilibrium between transmembrane and nontransmembrane helix topography and transmembrane helix positioning. Biochemistry 2004, 43:8794-8806. 50. Ulmschneider MB, Sansom MS, Di Nola A: Properties of integral membrane protein structures: derivation of an implicit membrane potential. Proteins 2005, 59:252-265. 51. Senes A, Chadi DC, Law PB, Walters RF, Nanda V, Degrado WF: E(z), a depth-dependent potential for assessing the energies of insertion of amino acid side-chains into membranes: derivation and applications to determining the orientation of transmembrane and interfacial helices. J Mol Biol 2007, 366:436-448. www.sciencedirect.com
61. Ren J, Lew S, Wang J, London E: Control of the transmembrane orientation and interhelical interactions within membranes by hydrophobic helix length. Biochemistry 1999, 38:5905-5912.
63. Killian JA: Hydrophobic mismatch between proteins and lipids in membranes. Biochim Biophys Acta 1998, 1376:401-415. 64. Andersen OS, Bruno MJ, Sun H, Koeppe RE 2nd: Singlemolecule methods for monitoring changes in bilayer elastic properties. Methods Mol Biol 2007, 400:543-570. 65. Nyholm TK, Ozdirekcan S, Killian JA: How protein transmembrane segments sense the lipid environment. Biochemistry 2007, 46:1457-1465. 66. Lerch-Bader M, Lundin C, Kim H, Nilsson I, von Heijne G: Contribution of positively charged flanking residues to the insertion of transmembrane helices into the endoplasmic reticulum. Proc Natl Acad Sci USA 2008, 105:4127-4132. 67. Meindl-Beinker NM, Lundin C, Nilsson I, White SH, von Heijne G: Asn- and Asp-mediated interactions between transmembrane helices during translocon-mediated membrane protein assembly. EMBO Rep 2006, 7:1111-1116. Current Opinion in Structural Biology 2009, 19:464–472
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68. Lew S, London E: Simple procedure for reversed-phase highperformance liquid chromatographic purification of long hydrophobic peptides that form transmembrane helices. Anal Biochem 1997, 251:113-116.
70. Aisenbrey C, Bechinger B, Grobner G: Macromolecular crowding at membrane interfaces: adsorption and alignment of membrane peptides. J Mol Biol 2008, 375:376-385.
69. Cheng HT, Megha, London E: Preparation and properties of asymmetric vesicles that mimic cell membranes: effect upon lipid raft formation and transmembrane helix orientation. J Biol Chem 2009, 284:6079-6092. This report describes the preparation of small unilamellar vesicles that mimic mammalian plasma membranes in the asymmetry of the lipid composition in their inner and outer leaflets. This may open the way for studies of hydrophobic helices in more realistic molde membranes.
71. Caputo GA, London E: Using a novel dual fluorescence quenching assay for measurement of tryptophan depth within lipid bilayers to determine hydrophobic alpha-helix locations within membranes. Biochemistry 2003, 42:3265-3274.
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72. Meijberg W, Booth PJ: The activation energy for insertion of transmembrane alpha-helices is dependent on membrane composition. J Mol Biol 2002, 319:839-853.
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Transmembrane vs. non-transmembrane hydrophobic helix topography in model and natural membranes Erwin London and Khurshida Shahidullah Experimental studies have begun to define how formation of transmembrane (TM) vs. non-TM hydrophobic helix topographies is controlled. It has been found that topography is very sensitive to sequence and lipid structure. Interestingly, there is a broad agreement between studies using artificial model membranes and natural membranes. These studies provide important insights into membrane protein insertion and function. Addresses Department of Biochemistry and Cell Biology, Stony Brook, NY 11794-5215, United States Corresponding author: London, Erwin ([email protected])
Current Opinion in Structural Biology 2009, 19:464–472 This review comes from a themed issue on Engineering and Design Edited by Bill DeGrado and Brian Kuhlman Available online 7th August 2009 0959-440X/$ – see front matter # 2009 Elsevier Ltd. All rights reserved. DOI 10.1016/j.sbi.2009.07.007
Topography and its biological significance The structure and function of the helical hydrophobic segments of membrane proteins are impacted by their interaction with the membrane bilayer. One approach to characterize how a hydrophobic sequence interacts with membranes is to define its topography in terms of the degree to which forms non-TM states (either dissolved in aqueous solution or bound to a membrane surface), or forms a membrane-spanning TM state (Figure 1A). Multiple substates can also exist, including co-existing TM states with different insertion boundaries (i.e. with different transverse positions) and membrane-bound non-TM states that vary in the extent that they penetrate the bilayer, or tilt relative to the surface. Much recent progress has been made characterizing these topographies with synthetic hydrophobic sequences and model membranes [1–3,4,5–10]. However, in nature membrane insertion usually occurs within the translocon complex. Recently, methods allowing the analysis of the sequence dependence of translocon-dependent TM insertion have been developed [11,12]. Topography studies should help answer fundamental questions concerning membrane protein insertion. How do sequence and lipid composition control topography? Current Opinion in Structural Biology 2009, 19:464–472
To what extent does insertion in artificial and natural membranes differ? Is the structure of a hydrophobic helix in natural membranes kinetically controlled by the translocon, or does it reflect the energetics of its interactions with lipid bilayers? With regard to these points it is important to note that even in a lipid bilayer equilibration between TM and non-TM states cannot always occur. When a hydrophobic sequence is bounded on both sides by large hydrophilic domains, equilibration requires membrane translocation of a large hydrophilic domain. This self-translocation is only possible for specialized proteins, for example, certain protein toxins, although it may occur under some other conditions for ordinary proteins [13]. TM/non-TM interconversion is more facile for isolated hydrophobic sequences bounded on one side by only a few hydrophilic residues, or hydrophilic helical hairpins with a short hydrophilic connecting loop (Figure 1B). Studies of the sequence and lipid dependence of topography should also provide insights into post-translational topography changes. These are known to occur in a number of cases. The helices of diphtheria toxin T domain convert from a membrane-bound non-TM state to a TM hairpin thought to be essential for membrane translocation of its A chain [14,15]. Similar behavior has been observed for colicins and proposed for Bcl family proteins [16]. TM/non-TM interconversion in these cases is generally coupled to large-scale conformational changes in other parts of the protein [17]. Post-translational TM insertion has also been reported for proteins with a helixtype hydrophobic anchoring (tail) sequences capped by a short hydrophilic sequence [18,19]. The switch of helixforming antimicrobial peptides from membrane-bound non-TM states to TM states can be crucial for their pore formation [20]. In other cases the prevention of the stable TM insertion of a hydrophobic sequence may be crucial for function [21,22]. There may be many as yet unidentified cases of TM/nonTM interconversion. Genomic analysis suggests considerable overlap in hydrophobicity between TM and soluble sequences [23,24]. However, the behavior of sequences in the translocon suggests low overlap in hydrophobicity between secreted sequences and TM helices [12]. Even so, where the energies of TM and non-TM states are similar, significant amounts of both TM and non-TM states with distinct conformations and functions may form, as in the case of the PrP (prion) protein, where conformationally distinct TM and non-TM states may have important consequences for pathogenesis [25]. www.sciencedirect.com
Transmembrane vs. non-transmembrane hydrophobic helix topography London and Shahidullah 465
Figure 1
TM/non-TM interconversion. Hydrophobic sequences are represented by gray rectangles. A. Equilibria between different states. Equilibria between different secondary structures can also occur, but are not illustrated. B. TM/non-TM equilibration can be blocked by large hydrophilic domains that cannot cross membranes (top), but not by very short hydrophilic sequences at the end of a hydrophobic helix (middle) or middle of a hydrophobic hairpin (bottom). C. TM helix position can be affected by anchoring. When anchoring is weak the distance of the residue in the center of a hydrophobic sequence (here Trp (W)) TM will be shallower than when anchoring is strong (right).
Finally, studies of topography can lead to the design of hydrophobic helices with biomedical applications. The pHLIP peptide, which converts from a soluble to TM state at low pH, is a tumor marker, inserting specifically into the membrane of tumor cells because of their acidic milieu [26].
Distinguishing transmembrane from nontransmembrane topography Hydrophobic helix topography in model membranes can be studied by NMR [1,8,27], neutron diffraction [28–30], attenuated total reflection Fourier transform infrared spectroscopy [31–33] and oriented circular dichroism [34–36]. The latter two methods do not require isotopic substitution, and have high sensitivity. Differential scanning calorimetry can also evaluate topography, as only the TM state strongly perturbs the gel to liquid disordered melting transition of a lipid bilayer [6,37,38]. This method should probe the average of peptide behavior above and below the melting transition. Fluorescence spectroscopy is another sensitive method widely used for topography studies [7,9,10]. The membrane location of a Trp (as gauged by lmax and/or fluorwww.sciencedirect.com
escence quenching) when placed at the center of a hydrophobic helix can distinguish TM topography (Trp deep) from non-TM topography (Trp shallow). Combining lmax and quenching distinguishes cases in which a Trp has an intermediate depth from that in which non-TM and TM populations co-exist [2]. (NMR also readily detects co-existing TM and non-TM populations [1].) One disadvantage of fluorescence is that Trp may perturb the behavior of a sequence. This can be largely subtracted out when the differences between two Trp-bearing helices are studied. Correcting for alteration of hydrophobicity by Trp, or demonstrating that a Trp does not alter function can also rule out perturbation effects [2,19]. In natural membranes translocon-controlled topography can be studied [11,12,38,39,40,41]. This allows rapid investigation of a wide variety of sequences in a biological milieu. In one approach, protection from proteolysis is used to identify TM topography [40]. In another approach a test hydrophobic sequence flanked by N-linked glycosylation sites is introduced into a chimeric protein and the topography of newly synthesized protein is quantified by the levels of single (TM), and double (non-TM) Current Opinion in Structural Biology 2009, 19:464–472
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glycosylation. This is evaluated on SDS-PAGE via differences in molecular weight and quantitative staining. Studies in natural and model membranes have different strengths. Translocon-induced insertion is likely to define topographies that are relevant in vivo, while lipid composition and pH can be varied and their effects studied most easily using model membranes. However, there has been recent progress in controlling natural membrane lipid composition [13]. The properties of non-TM states are also most easily defined in model membranes. Finally, model membranes may be useful for understanding changes in topography occurring after the release of a membrane protein from the translocon. To measure the sequence dependence of topography TM and non-TM states must co-exist in measurable amounts. This can be achieved by controlling the hydrophobicity of a host sequence into which residues of interest are substituted [11]. Another method is to vary pH using hydrophobic sequences containing an ionizable residue, which destabilizes the TM state when charged [32]. A third method is to vary bilayer width [2]. The TM state is destabilized when bilayer width exceeds hydrophobic helix length (i.e. conditions of negative mismatch).
TM hydrophobicity threshold in allhydrophobic helices Studies of translocon-induced membrane insertion and of topography in zwitterionic phosphatidylcholine (PC) model membranes show that polyAla sequences are generally not hydrophobic enough to form a stable TM state [6,42,43]. In translocon experiments, in one study 19 Ala long sequences did form predominantly (>50%) TM structure [40]. However, the SerPhe sequence following the Ala might have slightly aided the formation of a TM topography. In another study, the presence of 3–5 Leu in a 19 residue long hydrophobic sequence otherwise composed of Ala was necessary for 50% TM topography (i.e. equal levels of TM and non-TM states) [11]. An analysis of genomic data showed that all-Ala hydrophobic sequences cannot form a stable TM structure, and 1–2 Leu in such sequences are required to form a TM state [23]. (This analysis included multi-TM helix proteins, in which helix–helix interaction can stabilize the TM state (see below).) In PC model membranes, it was found that 7 Leu mixed with 15 Ala (with Leu all throughout the hydrophilic sequence, but also all on one face of the helix) were barely enough for TM insertion [37]. Studies of Lys-flanked hydrophobic sequences showed that a polyAla sequence with or without a few Leu forms a stable TM state in vesicles containing anionic lipids, but does not in vesicles composed solely of PC [44]. In another study, no Current Opinion in Structural Biology 2009, 19:464–472
significant difference between TM stability was found for an alternating LeuAla sequence and an equivalent composition with a segment of Leu followed by a segment of Ala [45]. However, an amphiphilic arrangement of Ala and Leu might help stabilize a surface-bound nonTM state relative to a TM state, similar to its effect on the surface state stability relative to an unbound state [46].
Effect of hydrophilic residues in hydrophobic helices Using the mammalian translocon system and a sequence in which each type of amino acid was placed in the middle of a 19 residue LeunAlam sequence, a complete ‘biological hydrophobicity’ scale was derived [11]. From most to least hydrophobic, an order of I,L > F,V > C,M > A > W > T > Y > G > S > N,H > P > Q > R > E,K > D was determined. A second study showed that the scale was not greatly affected by whether the N or C-terminus of the LeunAlam sequence faced the cytosol [47]. A similar (but not identical) order was also later found in the yeast translocon and YidC-dependent insertion in E. coli [38,39]. Biological hydrophobicity values were well correlated (r = 0.85) with those from octanol–water partitioning [11], a direct measure of amino acid residue hydrophobicity. This implies that hydrophobicity is the primary factor determining participation of residues in TM helices. Furthermore, biological hydrophobicity values parallel the relative abundance of residues that had been found in an early study of TM segments in single TM helix proteins [48]. This relationship is reinforced by the similarity (r = 0.96) between biological hydrophobicity and the TM tendency scale [23]. TM tendency is a measure of the tendency of a residue to favor TM insertion derived from the compositions of sequences in databases of soluble and TM segments, using a novel analysis yielding a near-theoretical limit of accuracy. Thus, biological hydrophobicity reflects properties that apply to more than just the simple sequences. Model membrane measurements of the effects of hydrophilic residues introduced into hydrophobic helices is less complete [1,5,49]. TM insertion in polyAla sequences containing three ‘guest’ residues was found to decrease in the order I > L > G > S [9]. This could reflect both altered membrane binding (K1 in Figure 1) and alteration of the TM/surface topography equilibrium (K2 in Figure 1). In a study using fully membrane-associated Leu-rich hydrophobic peptides containing two substitutions, TM topography decreased in the order L > G > S > P > K > D [2]. With single substitutions the TM state was more stable, but it could still be discerned that His, Pro, Asp and Lys destabilized TM topography more than Ala, Gly, Phe or Ser. Notice that the order of destabilization of the TM state in these studies mirrors that in the translocon-based studies. In www.sciencedirect.com
Transmembrane vs. non-transmembrane hydrophobic helix topography London and Shahidullah 467
a third study, four substitutions were introduced into peptides containing a mixture of Leu and Ala [32]. Most of the substitutions tested were relatively hydrophobic. It was found that Trp, and especially Tyr, destabilized the TM state, relative to a surface-bound state, much more than other hydrophobic residues, consistent with their known preference for the polar–non polar interface of the bilayer. Other results (e.g. weaker stabilization of the TM state by Val than by Ser or Ala) were surprising, and might have been affected by steric constraints when four substitutions are placed on the same face of a helix [32].
Effect of ionizable (‘charged’) residues in hydrophobic helices When charged, ionizable residues (Asp, Glu, Lys, Arg, His) in hydrophobic helices should destabilize the TM state most strongly. Model membrane studies showed a single Asp residue near the center of a hydrophobic sequence containing mostly Leu prevents formation of TM topography when Asp is charged, but not when it is uncharged [5,49]. Near pH 7, a single Lys in highly hydrophobic sequences did not prevent the formation of a TM topography, while with two Lys, TM topography was of borderline stability, and with more than two the TM state did not form. Unfortunately, it is not clear if the Lys was charged [2,8]. Another study showed four charged His are sufficient to destroy TM orientation in an alternating LeuAla hydrophobic sequence, but four uncharged His are not [1]. How much do uncharged ionizable residues destabilize TM topography? A clue comes from a study mainly designed to measure the relative tendency of hydrophilic residues to shift TM helix transverse position [4]. The effects of negative mismatch upon the stability of TM topography indicated that both uncharged Asp and uncharged His destabilized TM topography similarly to Asn or Gln, while uncharged Glu was more similar to Ser [4].
Effect of hydrophilic residue sequence position How the position of a specific residue within a hydrophobic sequence alters TM insertion was systematically analyzed in the translocon system [12,11]. A smooth dependence of the extent of TM helix topography vs. residue position was found, with hydrophilic residues destabilizing TM helix topography most strongly when at the center of a hydrophobic sequence (i.e. when located at the bilayer center). The position dependence was strong for the most highly hydrophilic residues, while a somewhat flatter, inverted position dependence was found for hydrophobic residues. As expected, Trp and Tyr promoted TM helix topography most when close to the edge of the hydrophobic sequence. Overall, the profiles were very similar to those defined by statistical analysis of the abundance of different residues vs. diswww.sciencedirect.com
tance from the center of a hydrophobic segment for membrane proteins of known structure [12,50,51]. It must be cautioned that a single hydrophilic residue in a TM helix can cause it to undergo a transverse shift in location in order to locate the hydrophilic residue close to the bilayer surface [4,52–55]. These studies showed that in the TM state the relationship between the position of a hydrophilic residue in a hydrophobic sequence and its membrane depth is not trivial, and depends on hydrophilic residue type. In other words, equivalent positions of different hydrophilic residues in a sequence do not mean they have equivalent membrane depths. Transverse shifts should tend to mask what would otherwise be larger changes in TM/non-TM equilibrium. One translocon study examined the effect of pairs of guest hydrophilic residues, symmetrically varying their position relative to the center of the hydrophobic sequence [11]. This should tend to lessen transverse shifts, which would move one hydrophilic residue toward the membrane surface but pull the other hydrophilic residue toward the bilayer center, largely canceling out any energetic advantage of transverse shift.
TM helix minimum length It is generally assumed that TM proteins require nearly 20 residues to span the membrane hydrocarbon core. However, TM helices with shorter hydrophobic segments form, and their behavior is of interest because of their potential role in length-dependent sorting of TM sequences between Golgi (which averages 15 residue TM sequences) and plasma membrane (18–20 residues) [10,56,57]. In both bacterial and eukaryotic membranes sequences with as few as 10 or less consecutive hydrophobic residues form TM segments [40,58,59]. A glycosylation study found that a C-terminal tail of 12 Leu residues was sufficient to act as a TM anchor for synaptobrevin2 [60]. Several model membrane studies demonstrated that strong negative mismatch prevented TM insertion of highly hydrophobic peptides [2,10,27,49]. Model membrane studies using protocols in which the TM and membrane-bound non-TM states were in equilibrium also found TM topography was destabilized by negative mismatch, with 11 highly hydrophobic residues having borderline TM stability in bilayers with a physiologically relevant width, that is, composed of dioleoyl PC (DOPC). A value of 11–12 residues was also found for the minimum continuous hydrophobic segment needed to form TM topography when charged residues interrupted long hydrophobic sequences [2,49]. Another study showed that 10 hydrophobic residues were too short to form a TM helix in DOPC bilayers [27]. A detailed recent study in model membrane vesicles found that in DOPC 50%TM topography required a Current Opinion in Structural Biology 2009, 19:464–472
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minimum length of 11–12 residues for Leu sequences and 13 residues for less hydrophobic alternating Leu–Ala sequences [45,61]. As expected, longer lengths were necessary to form TM states in thicker bilayers. Charged flanking residues increased the minimum TM-forming length relative to uncharged hydrophilic residues. On the basis of energetic considerations it was predicted that minimum TM-forming length would increase greatly and be more hydrophobicity-dependent for sequences even more weakly hydrophobic than alternating Leu–Ala. Similar conclusions were obtained in a translocon study [12]. For 50% TM topography, the minimum length was near 10 for all-Leu hydrophobic sequences, 13 for sequences that were nearly half Leu and half Ala, and much more for less hydrophobic sequences, rising to 25 or more for hydrophobic sequences composed only of Ala. Somewhat smaller numbers for each of these cases were obtained in an earlier study, in which the hydrophobic sequences contained an additional SerPhe at the C-terminal, but the overall pattern was the same [40]. The effect of negative mismatch was found to have similar affects on the stability of the TM state for hydrophobic antibiotic peptides of the ‘peptiabol’ class [62]. These molecules are rich in the unusual hydrophobic amino acid a-aminoisobutyric acid. The 15-residue peptaibol ampullosporin A only formed a TM helix in very thin bilayers, while the 20-residue peptaibol alamethicin formed a TM state in bilayers with a medium width. How are short TM helices accommodated in a membrane? Adjustment of local bilayer width is certainly one important mechanism [63–65]. Partial burial of flanking residues in the membrane and helix oligomerization are also likely to be important [63].
Effect of residues flanking the TM sequence upon topography Residues surrounding (‘flanking’) the ends of a hydrophobic sequence can also affect topography. If these residues are much more hydrophilic than those within the sequence, they are considered to be outside of the hydrophobic sequence. However, if they are modestly hydrophilic the definition of the ends of a TM sequence can be arbitrary. One can define the overall sequence as having a short, more highly hydrophobic segment or a longer and less hydrophobic segment. In any case, model membrane experiments described above showed that increased hydrophilicity of the residues at the ends of a short hydrophobic sequence destabilized TM topography [45]. In the translocon system stabilization of TM topography was observed for hydrophobic sequences having cationic sequences at their C-terminal flanking side, which locates in the cytofacial leaflet [66]. These effects were observed even when the cationic residues and hydrophobic helix were separated by 10–15 apparently disordered Gly and Pro rich sequences. This stabilCurrent Opinion in Structural Biology 2009, 19:464–472
ization of the TM state may partly reflect favorable electrostatic interactions with anionic lipids on the cytofacial leaflet (see below). Destabilization of TM topography was observed with anionic residues on an N-terminal flanking side of a hydrophobic helix, which locates in the exofacial leaflet.
Effect of helix–helix interaction upon topography Interactions between TM, and non-TM, helices should also affect the TM/non-TM equilibrium. Both Asn–Asn and Asp–Asp interactions have been studied in the translocon system, and a significant stabilization of the TM topography was observed [67]. No doubt such helix-helix interactions are a major factor stabilizing TM topography for helices of multi-TM helix proteins, in which hydrophilic residues are often present for functional reasons.
Lipid composition dependence of topography The effect of lipid composition upon hydrophobic sequence binding to model membranes (i.e. K1, Figure 1A) has been much studied. Anionic lipids strongly promote membrane binding of hydrophobic peptides and proteins containing cationic residues [9]. The effect of lipid composition upon the equilibrium between membrane-bound TM and non-TM states (i.e. K2, Figure 1A) has only been studied recently [44]. The presence of anionic lipids in model membranes (at as low as 10 mol%, within the physiological range) was found to strongly stabilize TM topography for hydrophobic helices flanked by cationic residues. That this effect was electrostatic (i.e. Coulombic) was confirmed using His-flanked peptides. Stabilization of the TM configuration by anionic lipid was only observed at pH values at which His was charged. A surprising difference between behavior in model membranes containing phosphatidylserine (PS) and phosphatidylglycerol (PG) was observed [44]. Although both lipids are anionic, the depth of a Trp at the center of a hydrophobic sequence was much deeper in PS-containing vesicles than in PG-containing vesicles. Either PS stabilized the TM configuration to a greater degree than PG, or PS anchored the TM state such that the Trp was in a more fixed position at the bilayer center (Figure 1C). Interestingly, circular dichroism showed significantly higher helix content in PS-containing vesicles than in those containing PG or PC. This may offer clues into differences in the function of different anionic lipids. The effect of different zwitterionic (i.e. PC or phosphatidylethanolamine) or uncharged lipids (i.e. cholesterol) on the equilibrium between the membrane-bound nonTM and TM states was small, at most [44]. However, in another study using lipids with long monounsaturated acyl chains, cholesterol was found to destabilize TM www.sciencedirect.com
Transmembrane vs. non-transmembrane hydrophobic helix topography London and Shahidullah 469
topography [68]. This could be due to acyl chain straightening by cholesterol increasing negative mismatch [68]. Tighter lipid packing in the presence of cholesterol may also be involved. Methods to prepare vesicles with asymmetric lipid compositions in their inner and outer leaflets were recently developed [69]. It was found that an asymmetric distribution of anionic lipids promoted the formation of TM topography by a cationic residue-flanked hydrophobic helix [69]. Asymmetric model membranes that mimic cell membranes may be useful for more precise studies of how lipid bilayer physical state controls TM helix behavior (and function), including studies of the effects of bilayer width and lipid packing in liquid ordered membrane domains (lipid rafts).
Issues complicating comparison of the stabilities of different topographies in translocon and model membrane-based experiments Several factors complicate comparison of model membrane and translocon experiments. The sequences studied in the translocon and model membrane systems have been similar, but not identical. Another difference is that isolated helices are used in model membranes and chimeric proteins in the translocon. Furthermore, translocon and model membrane studies have used different hydrophilic flanking residues. Comparison of translocon-induced and model membrane insertion is also complicated by differences in terms of secondary structures of the non-TM state, and whether it is exposed to aqueous solution, buried within a protein, or bound to the membrane surface. The difference between the TM and non-TM energies should be largest when the non-TM state is fully exposed to aqueous solution. This might help explain the smaller difference in the TM/nonTM dependence on hydrophobicity measured in yeast relative to that in mammalian membranes [11,39]. Further complicating interpretation differences in TM insertion, the stability of a surface-bound state can be affected by which residues point toward solution and which point toward the center of the lipid bilayer, and how deeply the surface-bound state is buried in the bilayer. Crowding on the membrane surface due to unfavorable peptide– peptide interactions [70] or strain arising from peptideinduced expansion of the leaflet into which insertion occurs could destabilize surface-bound non-TM states. None of these factors would affect a non-TM state in solution. Another crucial issue is whether experimental topography reflects an equilibrium. In most model membrane experiments, the peptides used are water insoluble, and to avoid irreversible aggregation in solution samples are prepared by mixing peptide and lipid before vesicle www.sciencedirect.com
formation. Demonstration that the TM state is in facile equilibrium with a membrane-bound non-TM state has been shown by reversibly altering the TM/non-TM balance by altering bilayer width (via binding and dissociation of decane [68,71] or pH [5,32,49]), using highly hydrophobic sequences with only a couple of charged flanking residues. However, the behavior of water-soluble Lysflanked Ala-rich peptides suggest they face a kinetic barrier to TM insertion when added to preformed membranes [72]. Further studies of what sequences allow or block posttranslational TM/non-TM equilibration is a high priority. This knowledge could be used in concert with mutagenesis to control TM/non-TM interconversion in intact proteins, and so investigate its function. Whether translocon-induced insertion represents an equilibrium is unclear, although its correlation with both hydrophobicity and model membrane behavior suggests this might be the case.
Why do translocon and model membrane systems give similar results? Given the issues above, the similarity between the insertion in natural and model membranes is remarkable. It may be due to interaction of potential TM segments with the lipid bilayer during their residence in the translocon [11]. It is also possible that the properties of the walls of the translocon ‘pore’ (which are depleted in ionizable residues, but not polar residues) mimic the lipid bilayer to a limited degree.
Acknowledgement This work was supported by NIH grant GM 48596.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1.
Bechinger B: Towards membrane protein design: pH-sensitive topology of histidine-containing polypeptides. J Mol Biol 1996, 263:768-775.
2.
Caputo GA, London E: Cumulative effects of amino acid substitutions and hydrophobic mismatch upon the transmembrane stability and conformation of hydrophobic alpha-helices. Biochemistry 2003, 42:3275-3285.
3.
de Planque MR, Killian JA: Protein-lipid interactions studied with designed transmembrane peptides: role of hydrophobic matching and interfacial anchoring. Mol Membr Biol 2003, 20:271-284.
4.
Krishnakumar SS, London E: The control of transmembrane helix transverse position in membranes by hydrophilic residues. J Mol Biol 2007, 374:1251-1269. Hydrophilic residues within TM helices can induce a transverse shift so that the hydrophilic residue locates closer to the membrane surface while TM structure is maintained. In this fluorescence study hydrophilic residues were ranked in terms of the magnitude of shifts they induced in model membranes. The stability of the TM state under conditions of negative mismatch was found to decrease in a fashion related to the degree to which the transverse shifts shortened the length of the membrane-spanning sequence. Current Opinion in Structural Biology 2009, 19:464–472
470 Engineering and Design
5.
Lew S, Ren J, London E: The effects of polar and/or ionizable residues in the core and flanking regions of hydrophobic helices on transmembrane conformation and oligomerization. Biochemistry 2000, 39:9632-9640.
6.
Lewis RN, Zhang YP, Hodges RS, Subczynski WK, Kusumi A, Flach CR, Mendelsohn R, McElhaney RN: A polyalanine-based peptide cannot form a stable transmembrane alpha-helix in fully hydrated phospholipid bilayers. Biochemistry 2001, 40:12103-12111.
7.
Ren J, Lew S, Wang Z, London E: Transmembrane orientation of hydrophobic alpha-helices is regulated both by the relationship of helix length to bilayer thickness and by the cholesterol concentration. Biochemistry 1997, 36:10213-10220.
8.
Vogt B, Ducarme P, Schinzel S, Brasseur R, Bechinger B: The topology of lysine-containing amphipathic peptides in bilayers by circular dichroism, solid-state NMR, and molecular modeling. Biophys J 2000, 79:2644-2656.
9.
Liu LP, Deber CM: Anionic phospholipids modulate peptide insertion into membranes. Biochemistry 1997, 36:5476-5482.
10. Webb RJ, East JM, Sharma RP, Lee AG: Hydrophobic mismatch and the incorporation of peptides into lipid bilayers: a possible mechanism for retention in the Golgi. Biochemistry 1998, 37:673-679. 11. Hessa T, Kim H, Bihlmaier K, Lundin C, Boekel J, Andersson H, Nilsson I, White SH, von Heijne G: Recognition of transmembrane helices by the endoplasmic reticulum translocon. Nature 2005, 433:377-381. 12. Hessa T, Meindl-Beinker NM, Bernsel A, Kim H, Sato Y, Lerch Bader M, Nilsson I, White SH, von Heijne G: Molecular code for transmembrane-helix recognition by the Sec61 translocon. Nature 2007, 450:1026-1030. Using chimeric proteins and glycosylation-based topography determination this report defined the affect of every amino acid on the amount of TM topography formation in mammalian membranes as a function of the position of the residue within a hydrophobic sequence. In addition, the effect of the length of sequences composed of Leu and Ala necessary to form a TM state was measured as a function of hydrophobicity, which was controlled by the relative content of these two amino acids.
diacylglycerol kinase epsilon in a cell membrane and its relationship to predictions from modeling. J Mol Biol 2008, 383:797-809. 22. Dalley JA, Bulleid NJ: The endoplasmic reticulum (ER) translocon can differentiate between hydrophobic sequences allowing signals for glycosylphosphatidylinositol anchor addition to be fully translocated into the ER lumen. J Biol Chem 2003, 278:51749-51757. 23. Zhao G, London E: An amino acid ‘transmembrane tendency’ scale that approaches the theoretical limit to accuracy for prediction of transmembrane helices: relationship to biological hydrophobicity. Protein Sci 2006, 15:1987-2001. 24. Cunningham F, Rath A, Johnson RM, Deber CM: Distinctions between hydrophobic helices in globular proteins and transmembrane segments as factors in protein sorting. J Biol Chem 2009, 284:5395-5402. 25. Hegde RS, Mastrianni JA, Scott MR, DeFea KA, Tremblay P, Torchia M, DeArmond SJ, Prusiner SB, Lingappa VR: A transmembrane form of the prion protein in neurodegenerative disease. Science 1998, 279:827-834. 26. Andreev OA, Dupuy AD, Segala M, Sandugu S, Serra DA, Chichester CO, Engelman DM, Reshetnyak YK: Mechanism and uses of a membrane peptide that targets tumors and other acidic tissues in vivo. Proc Natl Acad Sci USA 2007, 104:7893-7898. The artificial pHLIP peptide, derived from a TM sequence found in bacteriorhodopsin, forms a TM state only at low pH owing to the presence of Asp residues in its hydrophobic sequence. It was found to accumulate several-fold in mouse tumors, and owing to the low pH in the tumor tissue. Peptides with Lys or Asn in place of Asp, did not have this property. 27. Harzer U, Bechinger B: Alignment of lysine-anchored membrane peptides under conditions of hydrophobic mismatch: a CD, 15N and 31P solid-state NMR spectroscopy investigation. Biochemistry 2000, 39:13106-13114. 28. Han X, Mihailescu M, Hristova K: Neutron diffraction studies of fluid bilayers with transmembrane proteins: structural consequences of the achondroplasia mutation. Biophys J 2006, 91:3736-3747.
13. Bogdanov M, Xie J, Dowhan W: Lipid-protein interactions drive membrane protein topogenesis in accordance with the positive-inside rule. J Biol Chem 2008, 284:9637-9641.
29. Bradshaw JP, Darkes MJ, Harroun TA, Katsaras J, Epand RM: Oblique membrane insertion of viral fusion peptide probed by neutron diffraction. Biochemistry 2000, 39:6581-6585.
14. Kachel K, Ren J, Collier RJ, London E: Identifying transmembrane states and defining the membrane insertion boundaries of hydrophobic helices in membrane-inserted diphtheria toxin T domain. J Biol Chem 1998, 273:22950-22956.
30. Dante S, Hauss T, Dencher NA: Beta-amyloid 25 to 35 is intercalated in anionic and zwitterionic lipid membranes to different extents. Biophys J 2002, 83:2610-2616.
15. Wang Y, Malenbaum SE, Kachel K, Zhan H, Collier RJ, London E: Identification of shallow and deep membrane-penetrating forms of diphtheria toxin T domain that are regulated by protein concentration and bilayer width. J Biol Chem 1997, 272:25091-25098. 16. Antignani A, Youle RJ: How do Bax and Bak lead to permeabilization of the outer mitochondrial membrane? Curr Opin Cell Biol 2006, 18:685-689. 17. Wang J, Rosconi MP, London E: Topography of the hydrophilic helices of membrane-inserted diphtheria toxin T domain: TH1TH3 as a hydrophilic tether. Biochemistry 2006, 45:8124-8134. 18. Wattenberg B, Lithgow T: Targeting of C-terminal (tail)anchored proteins: understanding how cytoplasmic activities are anchored to intracellular membranes. Traffic 2001, 2:66-71. 19. Fujita K, Krishnakumar SS, Franco D, Paul AV, London E, Wimmer E: Membrane topography of the hydrophobic anchor sequence of poliovirus 3A and 3AB proteins and the functional effect of 3A/3AB membrane association upon RNA replication. Biochemistry 2007, 46:5185-5199. 20. Huang HW: Molecular mechanism of antimicrobial peptides: the origin of cooperativity. Biochim Biophys Acta 2006, 1758:1292-1302. 21. Decaffmeyer M, Shulga YV, Dicu AO, Thomas A, Truant R, Topham MK, Brasseur R, Epand RM: Determination of the topology of the hydrophobic segment of mammalian Current Opinion in Structural Biology 2009, 19:464–472
31. Bechinger B, Ruysschaert JM, Goormaghtigh E: Membrane helix orientation from linear dichroism of infrared attenuated total reflection spectra. Biophys J 1999, 76:552-563. 32. Aisenbrey C, Goormaghtigh E, Ruysschaert JM, Bechinger B: Translocation of amino acyl residues from the membrane interface to the hydrophobic core: thermodynamic model and experimental analysis using ATR-FTIR spectroscopy. Mol Membr Biol 2006, 23:363-374. 33. Aisenbrey C, Kinder R, Goormaghtigh E, Ruysschaert JM, Bechinger B: Interactions involved in the realignment of membrane-associated helices. An investigation using oriented solid-state NMR and attenuated total reflection Fourier transform infrared spectroscopies. J Biol Chem 2006, 281:7708-7716. 34. Wu Y, Huang HW, Olah GA: Method of oriented circular dichroism. Biophys J 1990, 57:797-806. 35. Mykhailiuk PK, Afonin S, Palamarchuk GV, Shishkin OV, Ulrich AS, Komarov IV: Synthesis of trifluoromethyl-substituted proline analogues as 19F NMR labels for peptides in the polyproline II conformation. Angew Chem Int Ed Engl 2008, 47:5765-5767. 36. Burck J, Roth S, Wadhwani P, Afonin S, Kanithasen N, Strandberg E, Ulrich AS: Conformation and membrane orientation of amphiphilic helical peptides by oriented circular dichroism. Biophys J 2008, 95:3872-3881. 37. Lewis RN, Liu F, Krivanek R, Rybar P, Hianik T, Flach CR, Mendelsohn R, Chen Y, Mant CT, Hodges RS et al.: Studies of the www.sciencedirect.com
Transmembrane vs. non-transmembrane hydrophobic helix topography London and Shahidullah 471
minimum hydrophobicity of alpha-helical peptides required to maintain a stable transmembrane association with phospholipid bilayer membranes. Biochemistry 2007, 46:1042-1054. 38. Xie K, Hessa T, Seppala S, Rapp M, von Heijne G, Dalbey RE: Features of transmembrane segments that promote the lateral release from the translocase into the lipid phase. Biochemistry 2007, 46:15153-15161. In this report, proteolysis was used to detect the effect of sequence upon TM insertion induced by the YidC ‘translocon’ of bacteria, which handles different proteins and is distinct from the SecYEG translocon. The latter is analogous to that in mammals. The sequence dependence of YidCdependent insertion was strikingly similar to that induced by the mammalian translocon.
This study used a statistical analysis of membrane proteins with known structures to derive an apparent energy vs. position of a residue introduced into a hydrophobic sequence, similar to that carried out in references [12,50]. When these ‘energies’ were used to predict the behavior of Lys flanking polyLeu sequences, behaviors similar to those previously measured in model membranes were found. 52. Monne M, Nilsson I, Johansson M, Elmhed N, von Heijne G: Positively and negatively charged residues have different effects on the position in the membrane of a model transmembrane helix. J Mol Biol 1998, 284:1177-1183. 53. Nilsson I, Saaf A, Whitley P, Gafvelin G, Waller C, von Heijne G: Proline-induced disruption of a transmembrane alpha-helix in its natural environment. J Mol Biol 1998, 284:1165-1175.
39. Hessa T, Reithinger JH, von Heijne G, Kim H: Analysis of the transmembrane helix integration in the endoplasmic reticulum in S. cerevisiae. J Mol Biol 2009, 386:1222-1228. In this report, glycosylation was used to detect the effect of sequence upon TM insertion induced by the yeast translocon. The affect of different residues upon TM insertion was similar to that induced by the mammalian translocon, but there were notable differences in detail. Most striking was a scaled-down dependence of topography upon hydrophobicity relative to that in mammals.
54. Braun P, von Heijne G: The aromatic residues Trp and Phe have different effects on the positioning of a transmembrane helix in the microsomal membrane. Biochemistry 1999, 38:9778-9782.
40. Kuroiwa T, Sakaguchi M, Mihara K, Omura T: Systematic analysis of stop-transfer sequence for microsomal membrane. J Biol Chem 1991, 266:9251-9255.
56. Bretscher MS, Munro S: Cholesterol and the Golgi apparatus. Science 1993, 261:1280-1281.
41. Hessa T, White SH, von Heijne G: Membrane insertion of a potassium-channel voltage sensor. Science 2005, 307:1427. 42. Bechinger B: Membrane insertion and orientation of polyalanine peptides: a (15)N solid-state NMR spectroscopy investigation. Biophys J 2001, 81:2251-2256. 43. Nilsson I, Johnson AE, von Heijne G: How hydrophobic is alanine? J Biol Chem 2003, 278:29389-29393.
55. Stefansson A, Armulik A, Nilsson I, von Heijne G, Johansson S: Determination of N- and C-terminal borders of the transmembrane domain of integrin subunits. J Biol Chem 2004, 279:21200-21205.
57. Munro S: An investigation of the role of transmembrane domains in Golgi protein retention. EMBO J 1995, 14:4695-4704. 58. Adams GA, Rose JK: Structural requirements of a membranespanning domain for protein anchoring and cell surface transport. Cell 1985, 41:1007-1015. 59. Chen H, Kendall DA: Artificial transmembrane segments. Requirements for stop transfer and polypeptide orientation. J Biol Chem 1995, 270:14115-14122.
44. Shahidullah K, London E: Effect of lipid composition on the topography of membrane-associated hydrophobic helices: stabilization of transmembrane topography by anionic lipids. J Mol Biol 2008, 379:704-718. This fluorescence study examined the effect of lipid composition upon the balance between TM and non-TM topographies in model membranes. Anionic lipids were found to stabilize TM topography for a wide variety of hydrophobic sequences with cationic flanking residues.
60. Whitley P, Grahn E, Kutay U, Rapoport TA, von Heijne G: A 12-residue-long polyleucine tail is sufficient to anchor synaptobrevin to the endoplasmic reticulum membrane. J Biol Chem 1996, 271:7583-7586.
45. Krishnakumar SS, London E: Effect of sequence hydrophobicity and bilayer width upon the minimum length required for the formation of transmembrane helices in membranes. J Mol Biol 2007, 374:671-687.
62. Salnikov ES, Friedrich H, Li X, Bertani P, Reismann S, Hertweck C, O’Neil JD, Raap J, Bechinger B: Structure and alignment of the membrane-associated peptaibols ampullosporin A and alamethicin by oriented 15N and 31P solid-state NMR spectroscopy. Biophys J 2009, 96:86-100. Using NMR methods, this study showed that the effect of negative mismatch upon the stability of the TM state apply to natural bacterial peptides containing aminoisobutyric acid. Unlike alamethicin, the short peptide ampullosporin A only had the ability to form a TM state in very thin bilayers. Additional experiments suggested that these molecules formed mixed 310/a-helices. The authors hypothesize that balance between TM and non-TM states could be closely related to the way that these peptides exert their antibiotic activities.
46. Fernandez-Vidal M, Jayasinghe S, Ladokhin AS, White SH: Folding amphipathic helices into membranes: amphiphilicity trumps hydrophobicity. J Mol Biol 2007, 370:459-470. 47. Lundin C, Kim H, Nilsson I, White SH, von Heijne G: Molecular code for protein insertion in the endoplasmic reticulum membrane is similar for N(in)-C(out) and N(out)-C(in) transmembrane helices. Proc Natl Acad Sci USA 2008, 105:15702-15707. 48. Landolt-Marticorena C, Williams KA, Deber CM, Reithmeier RA: Non-random distribution of amino acids in the transmembrane segments of human type I single span membrane proteins. J Mol Biol 1993, 229:602-608. 49. Caputo GA, London E: Position and ionization state of Asp in the core of membrane-inserted alpha helices control both the equilibrium between transmembrane and nontransmembrane helix topography and transmembrane helix positioning. Biochemistry 2004, 43:8794-8806. 50. Ulmschneider MB, Sansom MS, Di Nola A: Properties of integral membrane protein structures: derivation of an implicit membrane potential. Proteins 2005, 59:252-265. 51. Senes A, Chadi DC, Law PB, Walters RF, Nanda V, Degrado WF: E(z), a depth-dependent potential for assessing the energies of insertion of amino acid side-chains into membranes: derivation and applications to determining the orientation of transmembrane and interfacial helices. J Mol Biol 2007, 366:436-448. www.sciencedirect.com
61. Ren J, Lew S, Wang J, London E: Control of the transmembrane orientation and interhelical interactions within membranes by hydrophobic helix length. Biochemistry 1999, 38:5905-5912.
63. Killian JA: Hydrophobic mismatch between proteins and lipids in membranes. Biochim Biophys Acta 1998, 1376:401-415. 64. Andersen OS, Bruno MJ, Sun H, Koeppe RE 2nd: Singlemolecule methods for monitoring changes in bilayer elastic properties. Methods Mol Biol 2007, 400:543-570. 65. Nyholm TK, Ozdirekcan S, Killian JA: How protein transmembrane segments sense the lipid environment. Biochemistry 2007, 46:1457-1465. 66. Lerch-Bader M, Lundin C, Kim H, Nilsson I, von Heijne G: Contribution of positively charged flanking residues to the insertion of transmembrane helices into the endoplasmic reticulum. Proc Natl Acad Sci USA 2008, 105:4127-4132. 67. Meindl-Beinker NM, Lundin C, Nilsson I, White SH, von Heijne G: Asn- and Asp-mediated interactions between transmembrane helices during translocon-mediated membrane protein assembly. EMBO Rep 2006, 7:1111-1116. Current Opinion in Structural Biology 2009, 19:464–472
472 Engineering and Design
68. Lew S, London E: Simple procedure for reversed-phase highperformance liquid chromatographic purification of long hydrophobic peptides that form transmembrane helices. Anal Biochem 1997, 251:113-116.
70. Aisenbrey C, Bechinger B, Grobner G: Macromolecular crowding at membrane interfaces: adsorption and alignment of membrane peptides. J Mol Biol 2008, 375:376-385.
69. Cheng HT, Megha, London E: Preparation and properties of asymmetric vesicles that mimic cell membranes: effect upon lipid raft formation and transmembrane helix orientation. J Biol Chem 2009, 284:6079-6092. This report describes the preparation of small unilamellar vesicles that mimic mammalian plasma membranes in the asymmetry of the lipid composition in their inner and outer leaflets. This may open the way for studies of hydrophobic helices in more realistic molde membranes.
71. Caputo GA, London E: Using a novel dual fluorescence quenching assay for measurement of tryptophan depth within lipid bilayers to determine hydrophobic alpha-helix locations within membranes. Biochemistry 2003, 42:3265-3274.
Current Opinion in Structural Biology 2009, 19:464–472
72. Meijberg W, Booth PJ: The activation energy for insertion of transmembrane alpha-helices is dependent on membrane composition. J Mol Biol 2002, 319:839-853.
www.sciencedirect.com