Self-association of transmembrane alpha-helices in model membranes: importance of helix orientation and role of hydrophobic mismatch

Interactions between transmembrane helices play a key role in almost all cellular processes involving membrane proteins. We have investigated helix-helix interactions in lipid bilayers with synthetic tryptophan-flanked peptides that mimic the membrane spanning parts of membrane proteins. The peptides were functionalized with pyrene to allow the self-association of the helices to be monitored by pyrene fluorescence and Trp-pyrene fluorescence resonance energy transfer (FRET). Specific labeling of peptides at either their N or C terminus has shown that helix-helix association occurs almost exclusively between antiparallel helices. Furthermore, computer modeling suggested that antiparallel association arises primarily from the electrostatic interactions between alpha-helix backbone atoms. We propose that such interactions may provide a force for the preferentially antiparallel association of helices in polytopic membrane proteins. Helix-helix association was also found to depend on the lipid environment. In bilayers of dioleoylphosphatidylcholine, in which the hydrophobic length of the peptides approximately matched the bilayer thickness, association between the helices was found to require peptide/lipid ratios exceeding 1/25. Self-association of the helices was promoted by either increasing or decreasing the bilayer thickness, and by adding cholesterol. These results indicate that helix-helix association in membrane proteins can be promoted by unfavorable protein-lipid interactions.     

Transmembrane electrical currents of spin-labeled hydrophobic ions.

When spin-labeled phosphonium ions are rapidly mixed with phospholipid vesicles, time-dependent changes in the electron paramagnetic resonance spectrum of the spin label are observed. These changes are interpreted in terms of transmembrane transport of the hydrophobic ion, and simple analysis of the data at different membrane potentials is shown to give the binding constant of the ion to both membrane surfaces, the permeability, and current-voltage relationship for the vesicle membrane in the presence of the hydrophobic ion. These results establish the time resolution for methods using the phosphonium ion as a probe of time-dependent potentials across vesicle membranes, as well as provide fundamental information regarding the binding and transport of hydrophobic cations across bilayers. This latter point is significant in view of the fact that hydrophobic cations have not been well characterized in planar bilayers due to their weak binding and low conductance.     

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.     

Transmembrane transport of peptidoglycan precursors across model and bacterial membranes

Translocation of the peptidoglycan precursor Lipid II across the cytoplasmic membrane is a key step in bacterial cell wall synthesis, but hardly understood. Using NBD-labelled Lipid II, we showed by fluorescence and TLC assays that Lipid II transport does not occur spontaneously and is not induced by the presence of single spanning helical transmembrane peptides that facilitate transbilayer movement of membrane phospholipids. MurG catalysed synthesis of Lipid II from Lipid I in lipid vesicles also did not result in membrane translocation of Lipid II. These findings demonstrate that a specialized protein machinery is needed for transmembrane movement of Lipid II. In line with this, we could demonstrate Lipid II translocation in isolated Escherichia coli inner membrane vesicles and this transport could be uncoupled from the synthesis of Lipid II at low temperatures. The transport process appeared to be independent from an energy source (ATP or proton motive force). Additionally, our studies indicate that translocation of Lipid II is coupled to transglycosylation activity on the periplasmic side of the inner membrane.     

Transmembrane helices can induce domain formation in crowded model membranes

We studied compositionally heterogeneous multi-component model membranes comprised of saturated lipids, unsaturated lipids, cholesterol, and α-helical TM protein models using coarse-grained molecular dynamics simulations. Reducing the mismatch between the length of the saturated and unsaturated lipid tails reduced the driving force for segregation into liquid-ordered (l(o)) and liquid-disordered (l(d)) lipid domains. Cholesterol depletion had a similar effect, and binary lipid mixtures without cholesterol did not undergo large-scale phase separation under the simulation conditions. The phase-separating ternary dipalmitoyl-phosphatidylcholine (DPPC)/dilinoleoyl-PC (DLiPC)/cholesterol bilayer was found to segregate into l(o) and l(d) domains also in the presence of a high concentration of ΤΜ helices. The l(d) domain was highly crowded with TM helices (protein-to-lipid ratio ~1:5), slowing down lateral diffusion by a factor of 5-10 as compared to the dilute case, with anomalous (sub)-diffusion on the μs time scale. The membrane with the less strongly unsaturated palmitoyl-linoleoyl-PC instead of DLiPC, which in the absence of TM α-helices less strongly deviated from ideal mixing, could be brought closer to a miscibility critical point by introducing a high concentration of TM helices. Finally, the 7-TM protein bacteriorhodopsin was found to partition into the l(d) domains irrespective of hydrophobic matching. These results show that it is possible to directly study the lateral reorganization of lipids and proteins in compositionally heterogeneous and crowded model biomembranes with coarse-grained molecular dynamics simulations, a step toward simulations of realistic, compositionally complex cellular membranes. This article is part of a Special Issue entitled: Protein Folding in Membranes.     

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.     

Transmembrane topography and evolutionary conservation of synaptophysin

Synaptophysin is the major integral membrane protein of small synaptic vesicles. Its primary structure deduced from rat and human complementary DNA sequences predicts that synaptophysin contains four transmembrane regions and a carboxyl-terminal domain having a novel repetitive structure. To elucidate the transmembrane organization of this protein in the synaptic vesicle, five antipeptide antibodies were raised. The site-specific antibodies were used to map the cognate sequences to the cytoplasmic or intravesicular side of the synaptic vesicle membrane by determining the susceptibility of the epitopes to proteolysis. The results confirm a topographic model for synaptophysin in which the protein spans the vesicle membrane four times, with both the amino and carboxyl terminus being cytoplasmic. In addition, the evolutionary conservation of the synaptophysin domains was addressed as a function of their membrane localization. To this end the primary structure of bovine synaptophysin was determined. Sequence comparisons between bovine, rat, and human synaptophysin revealed that only the intravesicular loops showed a significant number of amino acid substitutions (22%), while the transmembrane regions and cytoplasmic sequences were highly conserved (3% substitutions). These results depict synaptophysin as a protein with multiple membrane spanning regions whose functional site is likely to reside in highly conserved intramembranous and cytoplasmic sequences.     

Interaction of polynucleotides with natural and model membranes.

Polynucleotides adsorb on natural and model phospholipid membranes in the presence of Mg2+-cations. Adsorption of nucleic acids on membranes results in a considerable change of their secondary structure. The presence of model phosphatidylcholine membranes greatly stimulates the rate of the synthesis of RNA by E. coli RNA-polymerase on DNA template.     

Transmembrane and water-soluble helix bundles display reverse patterns of surface roughness.

Amino acid exposure and surface roughness were calculated for 12 helices from three transmembrane alpha-helix bundles and 13 helices from seven water-soluble alpha-helix bundles. Transmembrane helix bundles have relatively rough surfaces exposed to the lipid bilayer hydrocarbon chains and relatively smooth surfaces along helix-helix interfaces. This pattern is the reverse of what occurs in water-soluble helix bundles, where relatively rough surfaces are at the helix-helix interfaces and relatively smooth surfaces are exposed to water. The relatively rough exposed surfaces and buried smooth surfaces of transmembrane helices are likely to contribute to the stability of transmembrane helical bundles in a phospholipid environment.     

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.     

Transmembrane topography of the nicotinic acetylcholine receptor delta subunit.

Current folding models for the nicotinic acetylcholine receptor (AChR) predict either four or five transmembrane segments per subunit. The N-terminus of each subunit is almost certainly extracellular. We have tested folding models by determining biochemically the cellular location of an intermolecular disulfide bridge thought to lie at the delta subunit C-terminus. Dimers of AChR linked through the delta-delta bridge were prepared from Torpedo marmorata and T.californica electric organ. The disulfide's accessibility to hydrophilic reductants was tested in a reconstituted vesicle system. In right-side-out vesicles (greater than 95% ACh binding sites outwards), the bridge was equally accessible whether or not vesicles had been disrupted by freeze--thawing or by detergents. Control experiments based on the rate of reduction of entrapped diphtheria toxin and measurements of radioactive reductant efflux demonstrated that the vesicles provide an adequate permeability barrier. In reconstituted vesicles containing AChR dimers in scrambled orientations, right-side-out dimers were reduced to monomers three times more rapidly than inside-out dimers, consistent with the measured rate of reductant permeation. These observations indicate that in reconstituted vesicles the delta-delta disulfide bridge lies in the same aqueous space as the ACh binding sites. They are most easily reconciled with folding models that propose an even number of transmembrane crossing per subunit.     

Transmembrane asymmetry and lateral domains in biological membranes

It is generally assumed that rafts exist in both the external and internal leaflets of the membrane, and that they overlap so that they are coupled functionally and structurally. However, the two monolayers of the plasma membrane of eukaryotic cells have different chemical compositions. This out-of-equilibrium situation is maintained by the activity of lipid translocases, which compensate for the slow spontaneous transverse diffusion of lipids. Thus rafts in the outer leaflet, corresponding to domains enriched in sphingomyelin and cholesterol, cannot be mirrored in the inner cytoplasmic leaflet. The extent to which lipids contribute to raft properties can be conveniently studied in giant unilamellar vesicles. In these, cholesterol can be seen to condense with saturated sphingolipids or phosphatidylcholine to form μm scale domains. However, such rafts fail to model biological rafts because they are symmetric, and because their membranes lack the mechanism that establishes this asymmetry, namely proteins. Biological rafts are in general of nm scale, and almost certainly differ in size and stability in inner and outer monolayers. Any coupling between rafts in the two leaflets, should it occur, is probably transient and dependent not upon the properties of lipids, but on transmembrane proteins within the rafts.     

Transmembrane helix structure, dynamics, and interactions: multi-nanosecond molecular dynamics simulations.

To probe the fundamentals of membrane/protein interactions, all-atom multi-nanosecond molecular dynamics simulations were conducted on a single transmembrane poly(32)alanine helix in a fully solvated dimyristoyphosphatidylcholine (DMPC) bilayer. The central 12 residues, which interact only with the lipid hydrocarbon chains, maintained a very stable helical structure. Helical regions extended beyond these central 12 residues, but interactions with the lipid fatty-acyl ester linkages, the lipid headgroups, and water molecules made the helix less stable in this region. The C and N termini, exposed largely to water, existed as random coils. As a whole, the helix tilted substantially, from perpendicular to the bilayer plane (0 degree) to a 30 degrees tilt. The helix experienced a bend at its middle, and the two halves of the helix at times assumed substantially different tilts. Frequent hydrogen bonding, of up to 0.7 ns in duration, occurred between peptide and lipid molecules. This resulted in correlated translational diffusion between the helix and a few lipid molecules. Because of the large variation in lipid conformation, the lipid environment of the peptide was not well defined in terms of "annular" lipids and on average consisted of 18 lipid molecules. When compared with a "neat" bilayer without peptide, no significant difference was seen in the bilayer thickness, lipid conformations or diffusion, or headgroup orientation. However, the lipid hydrocarbon chain order parameters showed a significant decrease in order, especially in those methylene groups closest to the headgroup.     

Transport of oligonucleotides across natural and model membranes

Oligo- and polynucleotides can not diffuse through lipid membrane, however they are taken up by eukaryotic cells by endocytosis mediated by the nucleic acid specific receptors. The compounds find some way to escape from endosomes and reach nucleic acids in both cell nucleus and cytoplasm. Oligonucleotides bind to a few cell surface proteins which take part in the virus-cell interaction and in the development of immune response. Interaction of nucleic acids with cell surface proteins may play a role in development of some pathologies. The biological role of this interaction is unclear. Efficient delivery of oligonucleotides into eukaryotic cells can be achieved in some conditions by natural mechanisms and by using artificial carriers-membrane vehicles and cationic polymer micelles.     

A molecular model for lipid-protein interaction in membranes: the role of hydrophobic mismatch.

The interaction free energy between a hydrophobic, transmembrane, protein and the surrounding lipid environment is calculated based on a microscopic model for lipid organization. The protein is treated as a rigid hydrophobic solute of thickness dP, embedded in a lipid bilayer of unperturbed thickness doL. The lipid chains in the immediate vicinity of the protein are assumed to adjust their length to that of the protein (e.g., they are stretched when dP > doL) in order to bridge over the lipid-protein hydrophobic mismatch (dP-doL). The bilayer's hydrophobic thickness is assumed to decay exponentially to its asymptotic, unperturbed, value. The lipid deformation free energy is represented as a sum of chain (hydrophobic core) and interfacial (head-group region) contributions. The chain contribution is calculated using a detailed molecular theory of chain packing statistics, which allows the calculation of conformational properties and thermodynamic functions (in a mean-field approximation) of the lipid tails. The tails are treated as single chain amphiphiles, modeled using the rotational isometric state scheme. The interfacial free energy is represented by a phenomenological expression, accounting for the opposing effects of head-group repulsions and hydrocarbon-water surface tension. The lipid deformation free energy delta F is calculated as a function of dP-doL. Most calculations are for C14 amphiphiles which, in the absence of a protein, pack at an average area per head-group ao approximately equal to 32 A2 (doL approximately 24.5 A), corresponding to the fluid state of the membrane. When dP = doL, delta F > 0 and is due entirely to the loss of conformational entropy experienced by the chains around the protein. When dP > doL, the interaction free energy is further increased due to the enhanced stretching of the tails. When dP < doL, chain flexibility (entropy) increases, but this contribution to delta F is overcounted by the increase in the interfacial free energy. Thus, delta F obtains a minimum at dP-doL approximately 0. These qualitative interpretations are supported by detailed numerical calculations of the various contributions to the interaction free energy, and of chain conformational properties. The range of the perturbation of lipid order extends typically over few molecular diameters. A rather detailed comparison of our approach to other models is provided in the discussion.     

Distribution of small hydrophobic molecules in membranes. I. Artificial lipid membranes]

A hydrophobic uncharged fluorescent probe of 4-dimethylaminochalcone (DMC) interacted with synthetic phospholipid membranes. Comparison of absorption spectra and fluorescence of DMC in the membranes and organic solvents shows that in the membranes the DMC molecules are located not in the hydrocarbon layer but in the polar regions near the surface. The probe is distributed regularly along the surface forming no dimers and clusters. Polar groups which surround the probe in the membrane are less mobile than the molecules of organic solvents at the same temperature. The evaluation shows that the relaxation time of polar groups in the probe environment is longer than 0.15-10(-9) sec. The DMC molecules may be located in different sites of the membrane surface, which seem to differ from one another in the mobility of polar groups.     

Study of the topography of cannabinoids in model membranes using X-ray diffraction

Small-angle X-ray diffraction was used to determine the topography of (-)-delta 8-tetrahydrocannabinol in partially hydrated dimyristoylphosphatidylcholine bilayers. Electron density profiles of lipid bilayers in the presence and absence of the cannabinoid were calculated using Fourier transform. Step-function equivalent profiles were then constructed to obtain the absolute electron density scale. We have compared the electron density profiles of the above preparations to determine the location of the drug molecule in the bilayer. By using (-)-5'-iodo-delta 8-tetrahydrocannabinol in parallel experiments, we were also able to locate the iodine atom in the bilayer and deduce the conformation of the cannabinoid side alkyl chain. All comparisons were made between different preparations having the same mesomorphic form and total period repeat distance. To achieve this, we have carried out X-ray diffraction experiments at various temperatures to cover the different mesomorphic phases and combined our data with the corresponding results from differential scanning calorimetry. Based on the results of this work and previous data on the orientation of the cannabinoid in model membranes, we concluded that the phenolic hydroxy group of the drug molecule exists near the carbonyl groups of DMPC and that the average position of the iodine atom is approx. 5.5 A from the center (terminal methyl region) of the DMPC bilayer. This requires the cannabinoid side-chain to assume an orientation parallel to the bilayer chains.