Motifs of serine and threonine can drive association of transmembrane helices

Known sequence motifs containing key glycine residues can drive the homo-oligomerization of transmembrane helices. To find other motifs, a randomized library of transmembrane interfaces was generated in which glycine was omitted. The TOXCAT system, which measures transmembrane helix association in the Escherichia coli inner membrane, was used to select high-affinity homo-oligomerizing sequences in this library. The two most frequently occurring motifs were SxxSSxxT and SxxxSSxxT. Isosteric mutations of any one of the serine and threonine residues to non-polar residues abolished oligomerization, indicating that the interaction between these positions is specific and requires an extended motif of serine and threonine hydroxyl groups. Computational modeling of these sequences produced several chemically plausible structures that contain multiple hydrogen bonds between the serine and threonine residues. While single serine or threonine side-chains do not appear to promote helix association, motifs can drive strong and specific association through a cooperative network of interhelical hydrogen bonds.     

The determinants of hydrophobic mismatch response for transmembrane helices

Hydrophobic mismatch arises from a difference in the hydrophobic thickness of a lipid membrane and a transmembrane protein segment, and is thought to play an important role in the folding, stability and function of membrane proteins. We have investigated the possible adaptations that lipid bilayers and transmembrane α-helices undergo in response to mismatch, using fully-atomistic molecular dynamics simulations totaling 1.4 μs. We have created 25 different tryptophan-alanine-leucine transmembrane α-helical peptide systems, each composed of a hydrophobic alanine–leucine stretch, flanked by 1–4 tryptophan side chains, as well as the β-helical peptide dimer, gramicidin A. Membrane responses to mismatch include changes in local bilayer thickness and lipid order, varying systematically with peptide length. Adding more flanking tryptophan side chains led to an increase in bilayer thinning for negatively mismatched peptides, though it was also associated with a spreading of the bilayer interface. Peptide tilting, bending and stretching were systematic, with tilting dominating the responses, with values of up to ~ 45° for the most positively mismatched peptides. Peptide responses were modulated by the number of tryptophan side chains due to their anchoring roles and distributions around the helices. Potential of mean force calculations for local membrane thickness changes, helix tilting, bending and stretching revealed that membrane deformation is the least energetically costly of all mismatch responses, except for positively mismatched peptides where helix tilting also contributes substantially. This comparison of energetic driving forces of mismatch responses allows for deeper insight into protein stability and conformational changes in lipid membranes.     

Polar residues in transmembrane helices can decrease electrophoretic mobility in polyacrylamide gels without causing helix dimerization

There are only a few available methods to study lateral interactions and self assembly of transmembrane helices. One of the most frequently used methods is sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) which can report on strong interactions between peptides in SDS solution. Here we offer a cautionary tale about studying the folding and assembly of membrane proteins using peptides and SDS-PAGE experiments as a membrane mimetic system. At least for the specific peptide and detergent systems studied here, we show that a polar asparagine residue in the 12th position of an otherwise hydrophobic helical segment of 20 amino acids causes a peptide to migrate on SDS-PAGE gels with an apparent molecular weight that is twice its true molecular weight, suggesting dimerization. However when examined carefully in SDS solutions and in situ in the polyacrylamide gel itself using Forster resonance energy transfer no interaction can be detected. Instead we show evidence suggesting that differential interactions between peptide and detergent drive the differences in electrophoretic mobility without any interaction between peptides. These results emphasize the need to apply multiple independent techniques to the study of membrane protein folding, and they highlight the usefulness of studying folding and structure of membrane proteins in lipid membranes rather than in detergents.     

Membrane insertion and dissociation processes of a model transmembrane helix

An important subject for elucidating membrane protein (MP) folding is how transmembrane helices (TMHs) insert into and dissociate from membranes. We investigated helix dissociation kinetics and insertion topology by means of intervesicular transfer of the fluorophore-labeled completely hydrophobic model transmembrane helix NBD-(LALAAAA)(3)-NH(2) (NBD = 7-nitro-2-1,3-benzoxadiazol-4-yl). The peptide forms a topologically stable transmembrane helix, which is in a monomer-antiparallel dimer equilibrium [Yano, Y., Takemoto, T., Kobayashi, S., Yasui, H., Sakurai, H., Ohashi, W., Niwa, M., Futaki, S., Sugiura, Y., and Matsuzaki, K. (2002) Biochemistry 41, 3073-3080]. The helix transfer kinetics, representing the helix dissociation process, was monitored by fluorescence recovery of the quenched peptide in donor vesicles containing a quencher upon its transfer to acceptor vesicles without the quencher. The transfer kinetics and vesicle concentration dependence demonstrated that the transfer was mediated by monomer in the aqueous phase. Furthermore, the activation enthalpy was estimated to be +17.7 +/- 1.3 kcal mol(-1). Helix insertion topology, detected by chemical quenching of the NBD group in the outer leaflet by dithionite ions, was found to be controlled by transmembrane electric potential-helix macro dipole interaction. On the basis of these observations, a model for the helix insertion/dissociation processes was discussed.     

The energetics of transmembrane helix insertion into a lipid bilayer

Free energy profiles for insertion of a hydrophobic transmembrane protein α-helix (M2 from CFTR) into a lipid bilayer have been calculated using coarse-grained molecular dynamics simulations and umbrella sampling to yield potentials of mean force along a reaction path corresponding to translation of a helix across a lipid bilayer. The calculated free energy of insertion is smaller when a bilayer with a thinner hydrophobic region is used. The free energies of insertion from the potentials of mean force are compared with those derived from a number of hydrophobicity scales and with those derived from translocon-mediated insertion. This comparison supports recent models of translocon-mediated insertion and in particular suggests that: 1), helices in an about-to-be-inserted state may be located in a hydrophobic region somewhat thinner than the core of a lipid bilayer; and/or 2), helices in a not-to-be-inserted state may experience an environment more akin (e.g., in polarity/hydrophobicity) to the bilayer/water interface than to bulk water.     

Modulation of the pHLIP transmembrane helix insertion pathway

The membrane-associated folding/unfolding of pH (low) insertion peptide (pHLIP) provides an opportunity to study how sequence variations influence the kinetics and pathway of peptide insertion into bilayers. Here, we present the results of steady-state and kinetics investigations of several pHLIP variants with different numbers of charged residues, with attached polar cargoes at the peptide's membrane-inserting end, and with three single-Trp variants placed at the beginning, middle, and end of the transmembrane helix. Each pHLIP variant exhibits a pH-dependent interaction with a lipid bilayer. Although the number of protonatable residues at the inserting end does not affect the ultimate formation of helical structure across a membrane, it correlates with the time for peptide insertion, the number of intermediate states on the folding pathway, and the rates of unfolding and exit. The presence of polar cargoes at the peptide's inserting end leads to the appearance of intermediate states on the insertion pathway. Cargo polarity correlates with a decrease of the insertion rate. We conclude that the existence of intermediate states on the folding and unfolding pathways is not mandatory and, in the simple case of a polypeptide with a noncharged and nonpolar inserting end, the folding and unfolding appears as an all-or-none transition. We propose a model for membrane-associated insertion/folding and exit/unfolding and discuss the importance of these observations for the design of new delivery agents for direct translocation of polar therapeutic and diagnostic cargo molecules across cellular membranes.     

Expression level of Sec62 modulates membrane insertion of marginally hydrophobic segments

In the endoplasmic reticulum (ER) membrane, transmembrane (TM) domain insertion occurs through the Sec61 channel with its auxiliary components, including Sec62. Sec62 interacts with the Sec61 channel and is located on the front side of the Sec61 lateral gate, an entry site for TM domains to the lipid bilayer. Overexpression of Sec62 led to a growth defect in yeast, and we investigated its effects on protein translocation and membrane insertion by pulse labeling of Sec62 client proteins. Our data show that the insertion efficiency of marginally hydrophobic TM segments is reduced upon Sec62 overexpression. This result suggests a potential regulatory role of Sec62 as a gatekeeper of the lateral gate, thereby modulating the insertion threshold of TM segments.     

Measurement of thermodynamic parameters for hydrophobic mismatch 1: self-association of a transmembrane helix

Membrane partitioning and self-association of transmembrane helices are crucial thermodynamic steps for membrane protein folding, although experimental difficulties have hampered quantitative estimations of related thermodynamic parameters, especially in lipid bilayer environments. This article reports for the first time, the complete set of thermodynamic parameters (DeltaG, DeltaH, DeltaS, and DeltaC(p)) for the formation of the antiparallel dimer of the inert hydrophobic model transmembrane helix X-(AALALAA)(3)-Y (X = 7-nitro-2-1, 3-benzoxadiazol-4-yl (NBD) and Y = NH(2) (I) or X = Ac and Y = NHCH(2)CH(2)-S-N-[4-[[4-(dimethylamino)phenyl]azo]phenyl]maleimide (DABMI) (II)) in dimonounsaturated phosphocholine lipid bilayers with different hydrophobic thicknesses (C14-C22) at 5-55 degrees C, as evaluated by fluorescence resonance energy transfer from I to II. Stronger dimerization was observed in thicker membranes and at lower temperatures (DeltaG = -9 to -26 kJ mol(-)(1)), driven by large negative DeltaH values (-18 to -80 kJ mol(-)(1)). Fourier transform infrared-polarized spectroscopy revealed that the peptide formed a stable transmembrane helix with an orientation angle of approximately 15 degrees in all bilayers without significant effects on lipid structures, suggesting that the depth to which the helix termini penetrate changes depending on the degree of hydrophobic mismatch. The enthalpy changes for helix-helix interactions can be well explained by the electrostatic interactions between helix macrodipoles in different dielectric environments. The new concept of dipole-dipole interaction as a basic driving force of helix dimerization will become a basis for understanding the structural and functional modifications in response to hydrophobic mismatch.     

Topological stability and self-association of a completely hydrophobic model transmembrane helix in lipid bilayers

Investigation of interactions between hydrophobic model peptides and lipid bilayers is perhaps the only way to elucidate the principles of the folding and stability of membrane proteins (White, S. H., and Wimley, W. C. (1998) Biochim. Biophys. Acta 1367, 339-352). We designed the completely hydrophobic "inert" peptide modeling a transmembrane (TM) helix without any of the specific side-chain interactions expected, X-(LALAAAA)(3)-NH(2) [X = Ac (I), 7-nitro-2-1,3-benzoxadiazol-4-yl (II), or 5(6)-carboxytetramethylrhodamine (III)]. Fourier transform infrared-polarized attenuated total reflection measurements revealed that I as well as II assume a TM helix in hydrated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayers. Dithionite quenching experiments detected no topological change (flip-flop) in the helix II for at least 24 h. Thus, the TM helix itself is a highly stable structure, even in the absence of flanking hydrophilic or aromatic amino acids which are suggested to play important roles in stable TM positioning. Helix self-association in lipid bilayers was detected by fluorescence resonance energy transfer between II and III. The peptide was in a monomer-antiparallel dimer equilibrium with an association free energy of approximately -13 kJ/mol. Electron spin resonance spectra of 1-palmitoyl-2-stearoyl-(14-doxyl)-sn-glycero-3-phosphocholine demonstrated the presence of a motionally restricted component at lower temperatures.     

Thermodynamics of insertion and self-association of a transmembrane helix: a lipophobic interaction by phosphatidylethanolamine

Thermodynamic parameters for the insertion and self-association of transmembrane helices are important for understanding the folding of helical membrane proteins. The lipid composition of bilayers would significantly affect these fundamental processes, although how is not well understood. Experimental systems using model transmembrane helices and lipid bilayers are useful for measuring and interpreting thermodynamic parameters (ΔG, ΔH, ΔS, and ΔC(p)) for the processes. In this study, the effect of the charge, phase, acyl chain unsaturation, and lateral pressure profile of bilayers on the membrane partitioning of the transmembrane helix (AALALAA)(3) was examined. Furthermore, the effect of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylethanolamine (POPE) on the thermodynamics for insertion and self-association of the helix in host membranes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) was investigated in detail. Interbilayer transfer of the helix monomer from POPC to POPC/POPE (1/1) bilayers was unfavorable (ΔG = +4.5 ± 2.9 kJ mol(-1) at 35 °C) due to an increase in enthalpy (ΔH = +31.1 ± 2.1 kJ mol(-1)). On the other hand, antiparallel dimerization of the helices in POPC/POPE (1/1) bilayers was enhanced compared with that in POPC bilayers (ΔΔG = -4.9 ± 0.2 kJ mol(-1) at 35 °C) due to a decrease in enthalpy (ΔΔH = -33.2 ± 1.5 kJ mol(-1)). A greater thickness of POPC/POPE bilayers only partially explained the observed effects. The residual effects could be related to changes in other physical properties such as higher lateral pressure in the hydrocarbon core in the PE-containing membrane. The origin of the enthalpy-driven "lipophobic" force that modulates the insertion and association of transmembrane helices will be discussed.     

Thermodynamic measurements of bilayer insertion of a single transmembrane helix chaperoned by fluorinated surfactants

Accurate determination of the free energy of transfer of a helical segment from an aqueous into a transmembrane (TM) conformation is essential for understanding and predicting the folding and stability of membrane proteins. Until recently, direct thermodynamically sound measurements of free energy of insertion of hydrophobic TM peptides were impossible due to peptide aggregation outside the lipid bilayer. Here, we overcome this problem by using fluorinated surfactants that are capable of preventing aggregation but, unlike detergents, do not themselves interact with the bilayer. We have applied the fluorescence correlation spectroscopy methodology to study surfactant-chaperoned insertion into preformed POPC (palmitoyloleoylphosphatidylcholine) vesicles of the two well-studied dye-labeled TM peptides of different lengths: WALP23 and WALP27. Extrapolation of the apparent free-energy values measured in the presence of surfactants to a zero surfactant concentration yielded free-energy values of -9.0±0.1 and -10.0±0.1 kcal/mol for insertion of WALP23 and WALP27, respectively. Circular dichroism measurements confirmed helical structure of peptides in lipid bilayer, in the presence of surfactants, and in aqueous mixtures of organic solvents. From a combination of thermodynamic and conformational measurements, we conclude that the partitioning of a four-residue L-A-L-A segment in the context of a continuous helical conformation from an aqueous environment into the hydrocarbon core of the membrane has a favorable free energy of 1 kcal/mol. Our measurements, combined with the predictions of two independent experimental hydrophobicity scales, indicate that the per-residue cost of transfer of the helical backbone from water to the hydrocarbon core of the lipid bilayer is unfavorable and is equal to +2.13±0.17 kcal/mol.     

Measurement of thermodynamic parameters for hydrophobic mismatch 2: intermembrane transfer of a transmembrane helix

Hydrophobic matching between proteins and lipids is essential for the thermodynamic stability of integral membrane proteins. However, there is no direct thermodynamic information available about the intermembrane transfer of proteins between membranes with different hydrophobic thicknesses, which is crucial for understanding hydrophobic mismatch. This article reports the complete set of thermodynamic parameters (DeltaG, DeltaH, DeltaS, and DeltaC(p)) for the intermembrane transfer of the inert hydrophobic model transmembrane helix NBD-(AALALAA)(3)-NH(2) (NBD: 7-nitro-2-1,3-benzoxadiazol-4-yl), which is exchangeable between vesicles, from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) to dimonounsaturated-phosphocholine lipid bilayers with different hydrophobic thicknesses (C14-C22) at 37-58 degrees C. The transfer free energies were calculated from equilibrium values of the extent of helix transfer from donor to acceptor lipid vesicles, as monitored by a decrease in fluorescence resonance energy transfer from the NBD group to a lipid-labeled Rhodamine in the donor upon transfer to the quencher-free acceptor. Under hydrophobic mismatch conditions up to approximately 7 A, the helix partitioning became unfavorable up to +7 kJ mol(-)(1), hampered by an increase in entropic (up to +20 kJ mol(-)(1)) and enthalpic (up to +66 kJ mol(-)(1)) terms in thinner and thicker membranes, respectively. Together with the results that H/D exchange at the membrane interface was accelerated in thinner membranes the obtained thermodynamic parameters were reasonably explained assuming that hydrophobic mismatch induces aqueous exposure or membrane burial of the helix termini, resulting in excess energies originating from the hydration of terminal hydrophobic residues or the unfavorable Born energy of terminal partial charges of the helix macrodipole.     

Effects of 'hydrophobic mismatch' on the location of transmembrane helices in the ER membrane

We have studied the effects of 'hydrophobic mismatch' between a poly-Leu transmembrane helix (TMH) and the ER membrane using a glycosylation mapping approach. The simplest interpretation of our results is that the lumenal end of the TMH is located deeper in the membrane for both short (negative mismatch) and long (positive mismatch) TMHs than for poly-Leu segments of intermediate length. We further find that the position-specific effect of Lys residues on the location of short TMHs in the membrane varies with an apparent helical periodicity when the Lys residue is moved along the poly-Leu stretch. We discuss these findings in the context of models for peptide-lipid interactions during hydrophobic mismatch.     

Regulation of G protein-coupled cAMP receptor activation by a hydrophobic residue in transmembrane helix 3

cAR1, a G protein-coupled cAMP receptor, is essential for multicellular development of Dictyostelium. We previously identified a cAR1-Ile(104) mutant that appeared to be constitutively activated based on its constitutive phosphorylation, elevated affinity for cAMP, and dominant-negative effects on development as well as specific cAR1 pathways that are subject to adaptation. To investigate how Ile(104) might regulate cAR1 activation, we assessed the consequences of substituting it with all other amino acids. Constitutive phosphorylation of these Ile(104) mutants varied broadly, suggesting that they are activated to varying extents, and was correlated with polarity of the substituting amino acid residue. Remarkably, all Ile(104) substitutions, except for the most conservative, dramatically elevated the receptor's cAMP affinity. However, only a third of the mutants (those with the most polar substitutions) blocked development. These findings are consistent with a model in which polar Ile(104) substitutions perturb the equilibrium between inactive and active cAR1 conformations in favour of the latter. Based on homology with rhodopsin, Ile(104) is likely buried within inactive cAR1 and exposed to the cytoplasm upon activation. We propose that the hydrophobic effect normally promotes burial of Ile(104) and hence cAR1 inactivation, while polar substitution of Ile(104) mitigates this effect, resulting in activation.     

Determining the membrane topology of proteins: insertion pathway of a transmembrane helix of annexin 12

We describe a sensitive method for determining the bilayer topology of single-site cysteine-linked NBD fluorescent labels on membrane proteins. Based upon a method developed for peptides [W. C. Wimley and S. H. White (2000) Biochemistry 39, 161-170], it utilizes a novel fluorescence quencher, lysoUB, comprised of a single acyl chain attached to a UniBlue chromophore. The enhanced sensitivity of the method arises from the brightness of the NBD fluorescence and the quenching efficiency of lysoUB, which is not fluorescent. In the course of validating the method, we examined the insertion topology of the D-E helical region of repeat 2 of annexin 12, known to adopt a transbilayer orientation at mildly acidic pH [Langen et al. (1998) Proc. Natl. Acad. Sci. USA 95, 14060-14065]. In the final membrane-inserted state, an NBD label attached to the single-cysteine mutant D134C was found to be in the outer (cis) leaflet, while the one attached to D162C was found in the trans leaflet. But kinetic measurements of NBD fluorescence suggested the existence of a transient intermediate insertion state whose lifetime could be increased by increasing the fraction of anionic lipids in the vesicles. Indeed, the lifetime could be increased for times sufficient for the completion of lysoUB-NBD topology measurements. Such measurements revealed that the D-E region adopts an interfacial topology in the intermediate state with both ends on the cis side of the membrane, consistent with the general concept of interface-directed membrane insertion of proteins [White et al. (2001) J. Biol. Chem. 276, 32395-32398].     

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.     

Control of the transmembrane orientation and interhelical interactions within membranes by hydrophobic helix length

We examined the effect of the length of the hydrophobic core of Lys-flanked poly(Leu) peptides on their behavior when inserted into model membranes. Peptide structure and membrane location were assessed by the fluorescence emission lambdamax of a Trp residue in the center of the peptide sequence, the quenching of Trp fluorescence by nitroxide-labeled lipids (parallax analysis), and circular dichroism. Peptides in which the hydrophobic core varied in length from 11 to 23 residues were found to be largely alpha-helical when inserted into the bilayer. In dioleoylphosphatidylcholine (diC18:1PC) bilayers, a peptide with a 19-residue hydrophobic core exhibited highly blue-shifted fluorescence, an indication of Trp location in a nonpolar environment, and quenching localized the Trp to the bilayer center, an indication of transmembrane structure. A peptide with an 11-residue hydrophobic core exhibited emission that was red-shifted, suggesting a more polar Trp environment, and quenching showed the Trp was significantly displaced from the bilayer center, indicating that this peptide formed a nontransmembranous structure. A peptide with a 23-residue hydrophobic core gave somewhat red-shifted fluorescence, but quenching demonstrated the Trp was still close to the bilayer center, consistent with a transmembrane structure. Analogous behavior was observed when the behavior of individual peptides was examined in model membranes with various bilayer widths. Other experiments demonstrated that in diC18:1PC bilayers the dilution of the membrane concentration of the peptide with a 23-residue hydrophobic core resulted in a blue shift of fluorescence, suggesting the red-shifted fluorescence at higher peptide concentrations was due to helix oligomerization. The intermolecular self-quenching of rhodamine observed when the peptide was rhodamine-labeled, and the concentration dependence of self-quenching, supported this conclusion. These studies indicate that the mismatch between helix length and bilayer width can control membrane location, orientation, and helix-helix interactions, and thus may mismatch control both membrane protein folding and the interactions between membrane proteins.     

Asparagine-mediated self-association of a model transmembrane helix

In membrane proteins, the extent to which polarity, hydrogen bonding, and van der Waals packing interactions of the buried, internal residues direct protein folding and association of transmembrane segments is poorly understood. The energetics associated with these various interactions should differ substantially between membrane versus water-soluble proteins. To help evaluate these energetics, we have altered a water-soluble, two-stranded coiled-coil peptide to render its sequence soluble in membranes. The membrane-soluble peptide associates in a monomer-dimer-trimer equilibrium, in which the trimer predominates at the highest peptide/detergent ratios. The oligomers are stabilized by a buried Asn side chain. Mutation of this Asn to Val essentially eliminates oligomerization of the membrane-soluble peptide. Thus, within a membrane-like environment, interactions involving a polar Asn side chain provide a strong thermodynamic driving force for membrane helix association.     

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.     

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.