Identification of the hydrophobic thickness of a membrane protein using fluorescence spectroscopy: studies with the mechanosensitive channel MscL

The hydrophobic thickness of a membrane protein is an important parameter, defining how the protein sits within the hydrocarbon core of the lipid bilayer that surrounds it in a membrane. Here we show that Trp scanning mutagenesis combined with fluorescence spectroscopy can be used to define the hydrophobic thickness of a membrane protein. The mechanosensitive channel of large conductance (MscL) contains two transmembrane alpha-helices, of which the second (TM2) is lipid-exposed. The region of TM2 that spans the hydrocarbon core of the bilayer when MscL is reconstituted into bilayers of dioleoylphosphatidylcholine runs from Leu-69 to Leu-92, giving a hydrophobic thickness of ca. 25 A. The results obtained using Trp scanning mutagenesis were confirmed using Cys residues labeled with the N-methyl-amino-7-nitroben-2-oxa-1,3-diazole [NBD] group; both fluorescence emission maxima and fluorescence lifetimes for the NBD group are sensitive to solvent dielectric constant over the range (2-40) thought to span the lipid headgroup region of a lipid bilayer. Changing phospholipid fatty acyl chain lengths from C14 and C24 results in no significant change for the fluorescence of the interfacial residues, suggesting very efficient hydrophobic matching between the protein and the surrounding lipid bilayer.     

The role of tryptophan residues in an integral membrane protein: diacylglycerol kinase

Tryptophan residues are thought to play special roles in integral membrane proteins, anchoring transmembrane alpha-helices into the lipid bilayer. We have studied the effect of mutating the five Trp residues in the diacylglycerol kinase (DGK) of Escherichia coli to Leu residues. The fluorescence emission maxima for DGK and a variety of Trp mutants in bilayers of dioleoylphosphatidylcholine [di(C18:1)PC] are all centered at ca. 327 nm, suggesting that all five Trp residues are located close to the glycerol backbone region of the bilayer. This is also consistent with fluorescence quenching experiments, measuring the separation between the Trp residues and the bromine atoms in a bilayer of dibromostearoylphosphatidylcholine. Mutation of Trp residues in DGK was found to have significant effects on activity for DGK reconstituted into bilayers of di(C18:1)PC containing 30 mol % 1,2-dihexanoylglycerol (DHG). Of the mutants containing a single Trp residue, only that containing Trp-112 was found to give active protein. The presence of both Trp-25 and Trp-112 gave higher activity than Trp-112 alone. Trp-25 and Trp-112 are the most important Trp residues in DGK as far as activity is concerned. Effects of mutations on K(m) for DHG were generally greater than effects on v(max). The activity of wild-type and mutant DHGs reconstituted into bilayers of phosphatidylcholines was sensitive to the chain length of the phospholipid, with highest activities for chain lengths of C18 or C20 and lower activities in phosphatidylcholines with shorter or longer chains. Compared to wild-type DGK, the Trp mutants were less affected by long-chain phosphatidylcholines but more affected by short-chain phospholipids. In mutants lacking Trp-25, low activities in short-chain phospholipids followed from a decrease in v(max) compared to wild type, combined with an increase in K(m) value for DHG, as observed in the wild type. It is suggested that Trp-25 plays a role in maintaining the alignment of ATP and DHG at the active site. Fluorescence emission spectra for the Trp mutants do not change significantly with changing fatty acyl chain length from C14 to C24, showing efficient hydrophobic matching between DGK and the surrounding lipid bilayer. It is suggested that hydrophobic matching is achieved by tilting of the transmembrane alpha-helix or rotation of residues at the ends of the helices about the Calpha-Cbeta bond linking the residue to the helix backbone. As well as any structural effects, the presence of Trp residues in DGK has a clear effect on thermal stability.     

Transmembrane domains of viral ion channel proteins: a molecular dynamics simulation study

Nanosecond molecular dynamics simulations in a fully solvated phospholipid bilayer have been performed on single transmembrane alpha-helices from three putative ion channel proteins encoded by viruses: NB (from influenza B), CM2 (from influenza C), and Vpu (from HIV-1). alpha-Helix stability is maintained within a core region of ca. 28 residues for each protein. Helix perturbations are due either to unfavorable interactions of hydrophobic residues with the lipid headgroups or to the need of the termini of short helices to extend into the surrounding interfacial environment in order to form H-bonds. The requirement of both ends of a helix to form favorable interactions with lipid headgroups and/or water may also lead to tilting and/or kinking of a transmembrane alpha-helix. Residues that are generally viewed as poor helix formers in aqueous solution (e.g., Gly, Ile, Val) do not destabilize helices, if located within a helix that spans a lipid bilayer. However, helix/bilayer mismatch such that a helix ends abruptly within the bilayer core destabilizes the end of the helix, especially in the presence of Gly and Ala residues. Hydrogen bonding of polar side-chains with the peptide backbone and with one another occurs when such residues are present within the bilayer core, thus minimizing the energetic cost of burying such side-chains.     

Membrane insertion of a voltage sensor helix

Most membrane proteins contain a transmembrane (TM) domain made up of a bundle of lipid-bilayer-spanning α-helices. TM α-helices are generally composed of a core of largely hydrophobic amino acids, with basic and aromatic amino acids at each end of the helix forming interactions with the lipid headgroups and water. In contrast, the S4 helix of ion channel voltage sensor (VS) domains contains four or five basic (largely arginine) side chains along its length and yet adopts a TM orientation as part of an independently stable VS domain. Multiscale molecular dynamics simulations are used to explore how a charged TM S4 α-helix may be stabilized in a lipid bilayer, which is of relevance in the context of mechanisms of translocon-mediated insertion of S4. Free-energy profiles for insertion of the S4 helix into a phospholipid bilayer suggest that it is thermodynamically favorable for S4 to insert from water to the center of the membrane, where the helix adopts a TM orientation. This is consistent with crystal structures of Kv channels, biophysical studies of isolated VS domains in lipid bilayers, and studies of translocon-mediated S4 helix insertion. Decomposition of the free-energy profiles reveals the underlying physical basis for TM stability, whereby the preference of the hydrophobic residues of S4 to enter the bilayer dominates over the free-energy penalty for inserting charged residues, accompanied by local distortion of the bilayer and penetration of waters. We show that the unique combination of charged and hydrophobic residues in S4 allows it to insert stably into the membrane.     

Revisiting Hydrophobic Mismatch with Free Energy Simulation Studies of Transmembrane Helix Tilt and Rotation

Protein-lipid interaction and bilayer regulation of membrane protein functions are largely controlled by the hydrophobic match between the transmembrane (TM) domain of membrane proteins and the surrounding lipid bilayer. To systematically characterize responses of a TM helix and lipid adaptations to a hydrophobic mismatch, we have performed a total of 5.8-μs umbrella sampling simulations and calculated the potentials of mean force (PMFs) as a function of TM helix tilt angle under various mismatch conditions. Single-pass TM peptides called WALPn (n = 16, 19, 23, and 27) were used in two lipid bilayers with different hydrophobic thicknesses to consider hydrophobic mismatch caused by either the TM length or the bilayer thickness. In addition, different flanking residues, such as alanine, lysine, and arginine, instead of tryptophan in WALP23 were used to examine their influence. The PMFs, their decomposition, and trajectory analysis demonstrate that 1), tilting of a single-pass TM helix is the major response to a hydrophobic mismatch; 2), TM helix tilting up to ∼10° is inherent due to the intrinsic entropic contribution arising from helix precession around the membrane normal even under a negative mismatch; 3), the favorable helix-lipid interaction provides additional driving forces for TM helix tilting under a positive mismatch; 4), the minimum-PMF tilt angle is generally located where there is the hydrophobic match and little lipid perturbation; 5), TM helix rotation is dependent on the specific helix-lipid interaction; and 6), anchoring residues at the hydrophilic/hydrophobic interface can be an important determinant of TM helix orientation.     

Membrane binding by MinD involves insertion of hydrophobic residues within the C-terminal amphipathic helix into the bilayer

MinD binds to phospholipid vesicles in the presence of ATP and is released by MinE, which stimulates the MinD ATPase. Membrane binding requires a short conserved C-terminal region, which has the potential to form an amphipathic helix. This finding has led to a model in which the binding of ATP regulates the formation or accessibility of this helix, which then embeds in the membrane bilayer. To test this model, we replaced each of the four hydrophobic residues within this potential helix with tryptophan or a charged residue. Introduction of a negatively charged amino acid decreased membrane binding of MinD and its ability to activate MinC. In contrast, mutants with tryptophan substitutions retained the ability to bind to the membrane and activate MinC. Fluorescence emission spectroscopy analysis of the tryptophan mutants F263W, L264W, and L267W confirmed that these tryptophan residues did insert into the hydrophobic interior of the bilayer. We conclude that membrane binding by MinD involves penetration of the hydrophobic residues within the C-terminal amphipathic helix into the hydrophobic interior of the bilayer.     

Effect of sequence hydrophobicity and bilayer width upon the minimum length required for the formation of transmembrane helices in membranes

The minimum hydrophobic length necessary to form a transmembrane (TM) helix in membranes was investigated using model membrane-inserted hydrophobic helices. The fluorescence of a Trp at the center of the sequence and its sensitivity to quenching were used to ascertain helix position within the membrane. Peptides with hydrophobic cores composed of poly(Leu) were compared to sequences containing a poly 1:1 Leu:Ala core (which have a hydrophobicity typical of natural TM helices). Studies varying bilayer width revealed that the poly(Leu) core peptides predominately formed a TM state when the bilayer width exceeded hydrophobic sequence length by (i.e. when negative mismatch was) up to ∼ 11-12 Å (e.g. the case of a 11-12 residue hydrophobic sequence in bilayers with a biologically relevant width, i.e. dioleoylphosphatidylcholine (DOPC) bilayers), while poly(LeuAla) core peptides formed predominantly TM state with negative mismatch of up to 9 Å (a 13 residue hydrophobic sequence in DOPC bilayers). This indicates that minimum length necessary to form a predominating amount of a TM state (minimum TM length) is only modestly hydrophobicity-dependent for the sequences studied here, and a formula that defines the minimum TM length as a function of hydrophobicity for moderately-to-highly hydrophobic sequences was derived. The minimum length able to form a stable TM helix for alternating LeuAla sequences, and that for sequences with a Leu block followed by an Ala block, was similar, suggesting that a hydrophobicity gradient along the sequence may not be an important factor in TM stability. TM stability was also similar for sequences flanked by different charged ionizable residues (Lys, His, Asp). However, ionizable flanking residues destabilized the TM configuration much more when charged than when uncharged. The ability of short hydrophobic sequences to form TM helices in membranes in the presence of substantial negative mismatch implies that lipid bilayers have a considerable ability to adjust to negative mismatch, and that short TM helices may be more common than generally believed. Factors that modulate the ability of bilayers to adjust to mismatch may strongly affect the configuration of short hydrophobic helices.     

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

The behavior of model-membrane-inserted polyLeu-rich peptides containing Asp residues located at various positions in their hydrophobic core was investigated. The topography of the bilayer-inserted alpha helices formed by these peptides was evaluated by measuring the emission lambda(max) and quenching the fluorescence of a Trp at the center of the peptide sequence. When Asp residues were protonated (at low pH), peptides that were incorporated into vesicles composed of dioleoylphosphatidylcholine (DOPC) adopted a topography in which the polyLeu sequence predominantly formed a normal transmembrane (TM) helix. When Asp residues were ionized (at neutral or high pH), topography was altered in a manner that would allow the charged Asp residues to reside near the bilayer surface. In DOPC vesicles, most peptides repositioned so that the longest segment of consecutive hydrophobic residues (12 residue minimum) formed a truncated/shifted TM structure. However, peptides with one or two charged Asp residues close to the center of the hydrophobic sequence and thus lacking even a 12-residue continuous hydrophobic segment, formed a helical non-TM state locating near the bilayer surface. At low pH, incorporation of the peptides into thicker bilayers composed of dierucoylphosphatidylcholine (DEuPC) resulted in the formation of a mixture of the normal TM state and the non-TM helical state located near the bilayer surface. In DEuPC vesicles at high pH, the non-TM state tended to predominate. How Asp-ionization-dependent shifts in helix topography may regulate the function of membrane proteins exposed to environments with differing pH in vivo (e.g., endosomes) is discussed.     

Structure of the membrane domain of subunit b of the Escherichia coli F0F1 ATP synthase.

The structure of the N-terminal transmembrane domain (residues 1-34) of subunit b of the Escherichia coli F0F1-ATP synthase has been solved by two-dimensional 1H NMR in a membrane mimetic solvent mixture of chloroform/methanol/H2O (4:4:1). Residues 4-22 form an alpha-helix, which is likely to span the hydrophobic domain of the lipid bilayer to anchor the largely hydrophilic subunit b in the membrane. The helical structure is interrupted by a rigid bend in the region of residues 23-26 with alpha-helical structure resuming at Pro-27 at an angle offset by 20 degrees from the transmembrane helix. In native subunit b, the hinge region and C-terminal alpha-helical segment would connect the transmembrane helix to the cytoplasmic domain. The transmembrane domains of the two subunit b in F0 were shown to be close to each other by cross-linking experiments in which single Cys were substituted for residues 2-21 of the native subunit and b-b dimer formation tested after oxidation with Cu(II)(phenanthroline)2. Cys residues that formed disulfide cross-links were found with a periodicity indicative of one face of an alpha-helix, over the span of residues 2-18, where Cys at positions 2, 6, and 10 formed dimers in highest yield. A model for the dimer is presented based upon the NMR structure and distance constraints from the cross-linking data. The transmembrane alpha-helices are positioned at a 23 degrees angle to each other with the side chains of Thr-6, Gln-10, Phe-14, and Phe-17 at the interface between subunits. The change in direction of helical packing at the hinge region may be important in the functional interaction of the cytoplasmic domains.     

Transmembrane helices of membrane proteins may flex to satisfy hydrophobic mismatch

A novel mechanism for membrane modulation of transmembrane protein structure, and consequently function, is suggested in which mismatch between the hydrophobic surface of the protein and the hydrophobic interior of the lipid bilayer induces a flexing or bending of a transmembrane segment of the protein. Studies on model hydrophobic transmembrane peptides predict that helices tilt to submerge the hydrophobic surface within the lipid bilayer to satisfy the hydrophobic effect if the helix length exceeds the bilayer width. The hydrophobic surface of transmembrane helix 1 (TM1) of lactose permease, LacY, is accessible to the bilayer, and too long to be accommodated in the hydrophobic portion of a typical lipid bilayer if oriented perpendicular to the membrane surface. Hence, nuclear magnetic resonance (NMR) data and molecular dynamics simulations show that TM1 from LacY may flex as well as tilt to satisfy the hydrophobic mismatch with the bilayer. In an analogous study of the hydrophobic mismatch of TM7 of bovine rhodopsin, similar flexing of the transmembrane segment near the conserved NPxxY sequence is observed. As a control, NMR data on TM5 of lacY, which is much shorter than TM1, show that TM5 is likely to tilt, but not flex, consistent with the close match between the extent of hydrophobic surface of the peptide and the hydrophobic thickness of the bilayer. These data suggest mechanisms by which the lipid bilayer in which the protein is embedded modulates conformation, and thus function, of integral membrane proteins through interactions with the hydrophobic transmembrane helices.     

Transmembrane helices of membrane proteins may flex to satisfy hydrophobic mismatch

A novel mechanism for membrane modulation of transmembrane protein structure, and consequently function, is suggested in which mismatch between the hydrophobic surface of the protein and the hydrophobic interior of the lipid bilayer induces a flexing or bending of a transmembrane segment of the protein. Studies on model hydrophobic transmembrane peptides predict that helices tilt to submerge the hydrophobic surface within the lipid bilayer to satisfy the hydrophobic effect if the helix length exceeds the bilayer width. The hydrophobic surface of transmembrane helix 1 (TM1) of lactose permease, LacY, is accessible to the bilayer, and too long to be accommodated in the hydrophobic portion of a typical lipid bilayer if oriented perpendicular to the membrane surface. Hence, nuclear magnetic resonance (NMR) data and molecular dynamics simulations show that TM1 from LacY may flex as well as tilt to satisfy the hydrophobic mismatch with the bilayer. In an analogous study of the hydrophobic mismatch of TM7 of bovine rhodopsin, similar flexing of the transmembrane segment near the conserved NPxxY sequence is observed. As a control, NMR data on TM5 of lacY, which is much shorter than TM1, show that TM5 is likely to tilt, but not flex, consistent with the close match between the extent of hydrophobic surface of the peptide and the hydrophobic thickness of the bilayer. These data suggest mechanisms by which the lipid bilayer in which the protein is embedded modulates conformation, and thus function, of integral membrane proteins through interactions with the hydrophobic transmembrane helices.     

Defining the transmembrane helix of M2 protein from influenza A by molecular dynamics simulations in a lipid bilayer

Integral membrane proteins containing at least one transmembrane (TM) alpha-helix are believed to account for between 20% and 30% of most genomes. There are several algorithms that accurately predict the number and position of TM helices within a membrane protein sequence. However, these methods tend to disagree over the beginning and end residues of TM helices, posing problems for subsequent modeling and simulation studies. Molecular dynamics (MD) simulations in an explicit lipid and water environment are used to help define the TM helix of the M2 protein from influenza A virus. Based on a comparison of the results of five different secondary structure prediction algorithms, three different helix lengths (an 18mer, a 26mer, and a 34mer) were simulated. Each simulation system contained 127 POPC molecules plus approximately 3500-4700 waters, giving a total of approximately 18,000-21,000 atoms. Two simulations, each of 2 ns duration, were run for the 18mer and 26mer, and five separate simulations were run for the 34mer, using different starting models generated by restrained in vacuo MD simulations. The total simulation time amounted to 11 ns. Analysis of the time-dependent secondary structure of the TM segments was used to define the regions that adopted a stable alpha-helical conformation throughout the simulation. This analysis indicates a core TM region of approximately 20 residues (from residue 22 to residue 43) that remained in an alpha-helical conformation. Analysis of atomic density profiles suggested that the 18mer helix revealed a local perturbation of the lipid bilayer. Polar side chains on either side of this region form relatively long-lived H-bonds to lipid headgroups and water molecules.     

Cumulative effects of amino acid substitutions and hydrophobic mismatch upon the transmembrane stability and conformation of hydrophobic alpha-helices

The effects of amino acid substitutions upon the behavior of poly(Leu)-rich alpha-helices inserted into model membrane vesicles were investigated. One or two consecutive Leu residues in the hydrophobic core of the helix were substituted with A, F, G, S, D, K, H, P, GG, SS, PG, PP, KK, or DD residues. A Trp placed at the center of the sequence allowed assessment of peptide behavior via fluorescence emission lambda(max) and dual quenching analysis of Trp depth [Caputo, G. A., and London, E. (2003) Biochemistry 42, 3265-3274]. In vesicles composed of dioleoylphosphatidylcholine (DOPC), all of the peptides with single substitutions adopted a transmembrane (TM) state. Experiments were also performed in thicker bilayers composed of dierucoylphosphatidylcholine (DEuPC). In DEuPC vesicles TM states were destabilized by mismatch between helix length and bilayer thickness. Nevertheless, in DEuPC vesicles TM states were still prevalent for peptides with single substitutions, although less so for peptides with P, K, H, or D substitutions. In contrast to single substitutions, certain consecutive double substitutions strongly interfered with formation of TM states. In both DOPC and DEuPC vesicles DD and KK substitutions abolished the normal TM state, but GG and SS substitutions had little effect. In even wider bilayers, a SS substitution reduced the formation of a TM state. A peptide with a PP substitution maintained the TM state in DOPC vesicles, but in DEuPC vesicles the level of formation of the TM state was significantly reduced. Upon disruption of normal TM insertion peptides moved close to the bilayer surface, with the exception of the KK-substituted peptide in DOPC vesicles, which formed a truncated TM segment. These studies begin to provide a detailed relationship between sequence and the stability of TM insertion and show that the influence of insertion-destabilizing residues upon hydrophobic helices can be strongly modulated by properties such as mismatch. For certain helix-forming hydrophobic sequences, sensitivity to lipid structure may be sufficient to induce large conformational changes in vivo.     

Ca2+ -ATPase structure in the E1 and E2 conformations: mechanism,helix-helix and helix-lipid interactions

The determination of the crystal structure of the Ca(2+)-ATPase of sarcoplasmic reticulum (SR) in its Ca(2+)-bound [Nature 405 (2000) 647] and Ca(2+)-free forms [Nature 418 (2002) 605] gives the opportunity for an analysis of conformational changes on the Ca(2+)-ATPase and of helix-helix and helix-lipid interactions in the transmembrane (TM) region of the ATPase. The locations of the ends of the TM alpha-helices on the cytoplasmic side of the membrane are reasonably well defined by the location of Trp residues and by the location of Lys-262 that snorkels up to the surface. The locations of the lumenal ends of the helices are less clear. The position of Lys-972 on the lumenal side of helix M9 suggests that the hydrophobic thickness of the protein is only about 21 A, rather than the normal 30 A. The experimentally determined TM alpha-helices do not agree well with those predicted theoretically. Charged headgroups are required for strong interaction of lipids with the ATPase, consistent with the large number of charged residues located close to the lipid-water interface. Helix packing appears to be rather irregular. Packing of helices M8 and M10 is of the 3-4 ridges-into-grooves or knobs-into-holes types. Packing of helices M5 and M7 involves two Gly residues in M7 and one Gly residue in M5. Packing of the other helices generally involves just one or two residues on each helix at the crossing point. The irregular packing of the TM alpha-helices in the Ca(2+)-ATPase, combined with the diffuse structure of the ATPase on the lumenal side of the membrane, is suggested to lead to a relative low activation energy for changing the packing of the TM alpha-helices, with changes in TM alpha-helical packing being important in the process of transfer of Ca(2+) ions across the membrane. The inhibitor thapsigargin binds in a cleft between TM alpha-helices M3, M5 and M7. It is suggested that this and other similar clefts provide binding sites for a variety of hydrophobic molecules affecting the activity of the Ca(2+)-ATPase.     

Structure of the integrin beta3 transmembrane segment in phospholipid bicelles and detergent micelles

Integrin adhesion receptors transduce bidirectional signals across the plasma membrane, with the integrin transmembrane domains acting as conduits in this process. Here, we report the first high-resolution structure of an integrin transmembrane domain. To assess the influence of the membrane model system, structure determinations of the beta3 integrin transmembrane segment and flanking sequences were carried out in both phospholipid bicelles and detergent micelles. In bicelles, a 30-residue linear alpha-helix, encompassing residues I693-H772, is adopted, of which I693-I721 appear embedded in the hydrophobic bicelle core. This relatively long transmembrane helix implies a pronounced helix tilt within a typical lipid bilayer, which facilitates the snorkeling of K716's charged side chain out of the lipid core while simultaneously immersing hydrophobic L717-I721 in the membrane. A shortening of bicelle lipid hydrocarbon tails does not lead to the transfer of L717-I721 into the aqueous phase, suggesting that the reported embedding represents the preferred beta3 state. The nature of the lipid headgroup affected only the intracellular part of the transmembrane helix, indicating that an asymmetric lipid distribution is not required for studying the beta3 transmembrane segment. In the micelle, residues L717-I721 are also embedded but deviate from linear alpha-helical conformation in contrast to I693-K716, which closely resemble the bicelle structure.     

Helical integrity and microsolvation of transmembrane domains from Flaviviridae envelope glycoproteins

Charged and polar amino acids in the transmembrane domains of integral membrane proteins can be crucial for protein function and also promote helix-helix association or protein oligomerization. Yet, our current understanding is still limited on how these hydrophilic amino acids are efficiently translocated from the Sec61/SecY translocon into the cell membrane during the biogenesis of membrane proteins. In hepatitis C virus, the putative transmembrane segments of envelope glycoproteins E1 and E2 were suggested to heterodimerize via a Lys-Asp ion-pair in the host endoplasmic reticulum. Therefore in this work, we carried out molecular dynamic simulations in explicit lipid bilayer and solvent environment to explore the stability of all possible bridging ion-pairs using the model of H-segment helix dimers. We observed that, frequently, several water molecules penetrated from the interface into the membrane core to stabilize the charged and polar pairs. The hydration time and amount of water molecules in the membrane core depended on the position of the charged residues as well as on the type of ion-pairs. Similar microsolvation events were observed in simulations of the putative E1-E2 transmembrane helix dimers. Simulations of helix monomers from other members of the Flaviviridae family suggest that these systems show similar behaviors. Thus this study illustrates the important contribution of water microsolvation to overcome the unfavorable energetic cost of burying charged and polar amino acids in membrane lipid bilayers. Also, it emphasizes the novel role of bridging charged or polar interactions stabilized by water molecules in the hydrophobic lipid bilayer core that has an important biological function for helix dimerization in several envelope glycoproteins from the family of Flaviviridae viruses.     

Topography of helices 5-7 in membrane-inserted diphtheria toxin T domain: identification and insertion boundaries of two hydrophobic sequences that do not form a stable transmembrane hairpin

The T domain of diphtheria toxin undergoes a low pH-induced conformational change that allows it to penetrate cell membranes. T domain hydrophobic helices 8 and 9 can adopt two conformations, one close to the membrane surface (P state) and a second in which they apparently form a transmembrane hairpin (TM state). We have now studied T domain helices 5-7, a second cluster of hydrophobic helices, using Cys-scanning mutagenesis. After fluorescently labeling a series of Cys residues, penetration into a non-polar environment, accessibility to externally added antibodies, and relative depth in the bilayer were monitored. It was found that helices 5-7 insert shallowly in the P state and deeply in the TM state. Thus, the conformational changes in helices 5-7 are both similar and somehow linked to those in helices 8 and 9. The boundaries of deeply inserting sequences were also identified. One deeply inserted segment was found to span residues 270 to 290, which overlaps helix 5, and a second spanned residues 300 to 320, which includes most of helix 6 and all of helix 7. This indicates that helices 6 and 7 form a continuous hydrophobic segment despite their separation by a Pro-containing kink. Additionally, it is found that in the TM state some residues in the hydrophilic loop between helices 5 and 6 become more highly exposed than they are in the P state. Their exposure to external solution in the TM state indicates that helices 5-7 do not form a stable transmembrane hairpin. However, helix 5 and/or helices 6 plus 7 could form transmembrane structures that are in equilibrium with non-transmembrane states, or be kinetically prevented from forming a transmembrane structure. How helices 5-7 might influence the mechanism by which the T domain aids translocation of the diphtheria toxin A chain across membranes is discussed.     

Proline-induced distortions of transmembrane helices

Proline residues in the transmembrane (TM) alpha-helices of integral membrane proteins have long been suspected to play a key role for helix packing and signal transduction by inducing regions of helix distortion and/or dynamic flexibility (hinges). In this study we try to characterise the effect of proline on the geometric properties of TM alpha-helices. We have examined 199 transmembrane alpha-helices from polytopic membrane proteins of known structure. After examining the location of proline residues within the amino acid sequences of TM helices, we estimated the helix axes either side of a hinge and hence identified a hinge residue. This enabled us to calculate helix kink and swivel angles. The results of this analysis show that proline residues occur with a significant concentration in the centre of sequences of TM alpha-helices. In this location, they may induce formation of molecular hinges, located on average about four residues N-terminal to the proline residue. A superposition of proline-containing TM helices structures shows that the distortion induced is anisotropic and favours certain relative orientations (defined by helix kink and swivel angles) of the two helix segments.     

Structure in the channel forming domain of colicin E1 bound to membranes: the 402-424 sequence

To explore the structure of the pore-forming fragment of colicin E1 in membranes, a series of 23 consecutive single cysteine substitution mutants was prepared in the sequence 402-424. Each mutant was reacted with a sulfhydryl-specific reagent to generate a nitroxide labeled side chain, and the mobility of the side chain and its accessibility to collision with paramagnetic reagents was determined from the electron paramagnetic resonance spectrum. Individual values of these quantities were used to identify tertiary contact sites and the nature of the surrounding solvent, while their periodic dependence on sequence position was used to identify secondary structure. In solution, the data revealed a regular helix of 11 residues in the region 406-416, consistent with helix IV of the crystal structure. Upon binding to negatively charged membranes at pH 4.0, helix IV apparently grows to a length of 19 residues, extending from 402-420. One face of the helix is solvated by the lipid bilayer, and the other by an environment of a polar nature. Surprisingly, a conserved charged pair, D408-R409, is located on the lipid-exposed face. Evidence is presented to suggest a transmembrane orientation of this new helix, although other topographies may exist in equilibrium.     

Transmembrane helix dynamics of bacterial chemoreceptors supports a piston model of signalling

Transmembrane α-helices play a key role in many receptors, transmitting a signal from one side to the other of the lipid bilayer membrane. Bacterial chemoreceptors are one of the best studied such systems, with a wealth of biophysical and mutational data indicating a key role for the TM2 helix in signalling. In particular, aromatic (Trp and Tyr) and basic (Arg) residues help to lock α-helices into a membrane. Mutants in TM2 of E. coli Tar and related chemoreceptors involving these residues implicate changes in helix location and/or orientation in signalling. We have investigated the detailed structural basis of this via high throughput coarse-grained molecular dynamics (CG-MD) of Tar TM2 and its mutants in lipid bilayers. We focus on the position (shift) and orientation (tilt, rotation) of TM2 relative to the bilayer and how these are perturbed in mutants relative to the wildtype. The simulations reveal a clear correlation between small (ca. 1.5 Å) shift in position of TM2 along the bilayer normal and downstream changes in signalling activity. Weaker correlations are seen with helix tilt, and little/none between signalling and helix twist. This analysis of relatively subtle changes was only possible because the high throughput simulation method allowed us to run large (n = 100) ensembles for substantial numbers of different helix sequences, amounting to ca. 2000 simulations in total. Overall, this analysis supports a swinging-piston model of transmembrane signalling by Tar and related chemoreceptors.