Search

Hydrophobic membrane-spanning helices often are flanked by interfacial aromatic or charged residues. In this paper, we compare the consequences of single Trp → Tyr substitutions at each interface for the properties of a defined transmembrane helix in the absence of charged residues. The choice of molecular framework is critical for these single-residue experiments because the presence of "too many" aromatic residues (more than one at either membrane-water interface) introduces excess dynamic averaging of solid state NMR observables. To this end, we compare the outcomes when changing W(5) or W(19), or both of them, to tyrosine in the well-characterized transmembrane peptide acetyl-GGALW(5)(LA)6LW(19)LAGA-amide ("GWALP23"). By means of solid-state (2)H and (15)N NMR experiments, we find that Y(19)GW(5)ALP23 displays similar magnitudes of peptide helix tilt as Y(5)GW(19)ALP23 and responds similarly to changes in bilayer thickness, from DLPC to DMPC to DOPC. The presence of Y(19) changes the azimuthal rotation angle ρ (about the helix axis) to a similar extent as Y(5), but in the opposite direction. When tyrosines are substituted for both tryptophans to yield GY(5,19)ALP23, the helix tilt angle is again of comparable magnitude, and furthermore, the preferred azimuthal rotation angle ρ is relatively unchanged from that of GW(5,19)ALP23. The extent of dynamic averaging increases marginally when Tyr replaces Trp. Yet, importantly, all members of the peptide family having single Tyr or Trp residues near each interface exhibit only moderate and not highly extensive dynamic averaging. The results provide important benchmarks for evaluating conformational and dynamic control of membrane protein function.

Membrane proteins are regulated by the lipid bilayer composition. Specific lipid-protein interactions rarely are involved, which suggests that the regulation is due to changes in some general bilayer property (or properties). The hydrophobic coupling between a membrane-spanning protein and the surrounding bilayer means that protein conformational changes may be associated with a reversible, local bilayer deformation. Lipid bilayers are elastic bodies, and the energetic cost of the bilayer deformation contributes to the total energetic cost of the protein conformational change. The energetics and kinetics of the protein conformational changes therefore will be regulated by the bilayer elasticity, which is determined by the lipid composition. This hydrophobic coupling mechanism has been studied extensively in gramicidin channels, where the channel-bilayer hydrophobic interactions link a 'conformational' change (the monomer [left and right arrow] dimer transition) to an elastic bilayer deformation. Gramicidin channels thus are regulated by the lipid bilayer elastic properties (thickness, monolayer equilibrium curvature, and compression and bending moduli). To investigate whether this hydrophobic coupling mechanism could be a general mechanism regulating membrane protein function, we examined whether voltage-dependent skeletal-muscle sodium channels, expressed in HEK293 cells, are regulated by bilayer elasticity, as monitored using gramicidin A (gA) channels. Nonphysiological amphiphiles ([beta]-octyl-glucoside, Genapol X-100, Triton X-100, and reduced Triton X-100) that make lipid bilayers less 'stiff', as measured using gA channels, shift the voltage dependence of sodium channel inactivation toward more hyperpolarized potentials. At low amphiphile concentration, the magnitude of the shift is linearly correlated to the change in gA channel lifetime. Cholesterol-depletion, which also reduces bilayer stiffness, causes a similar shift in sodium channel inactivation. These results provide strong support for the notion that bilayer-protein hydrophobic coupling allows the bilayer elastic properties to regulate membrane protein function. KEY WORDS: gramicidin A * bilayer material properties * bilayer deformation energy * hydrophobic coupling * lipid-protein interactions

The hydrophobic coupling between membrane-spanning proteins and the lipid bilayer core causes the bilayer thickness to vary locally as proteins and other 'defects' are embedded in the bilayer. These bilayer deformations incur an energetic cost that, in principle, could couple membrane proteins to each other, causing them to associate in the plane of the membrane and thereby coupling them functionally. We demonstrate the existence of such bilayer-mediated coupling at the single-molecule level using single-barreled as well as double-barreled gramicidin channels in which two gramicidin subunits are covalently linked by a water-soluble, flexible linker. When a covalently attached pair of gramicidin subunits associates with a second attached pair to form a double-barreled channel, the lifetime of both channels in the assembly increases from hundreds of milliseconds to a hundred seconds--and the conductance of each channel in the side-by-side pair is almost 10% higher than the conductance of the corresponding single-barreled channels. The double-barreled channels are stabilized some 100,000-fold relative to their single-barreled counterparts. This stabilization arises from: first, the local increase in monomer concentration around a single-barreled channel formed by two covalently linked gramicidins, which increases the rate of double-barreled channel formation; and second, from the increased lifetime of the double-barreled channels. The latter result suggests that the two barrels of the construct associate laterally. The underlying cause for this lateral association most likely is the bilayer deformation energy associated with channel formation. More generally, the results suggest that the mechanical properties of the host bilayer may cause the kinetics of membrane protein conformational transitions to depend on the conformational states of the neighboring proteins. KEY WORDS: gramicidin channels * bilayer mechanics * hydrophobic coupling * lateral association

Research has been conducted on the aromatic and positively charged side chains. The effect of these side chains on the lipid-peptide interactions has been investigated and the details are reported.

The extent of incorporation of membrane proteins is reduced when there is a hydrophobic mismatch between membrane lipids and membrane proteins. As the mismatch increases, smaller amounts of the protein are incorporated into the lipid bilayer and larger amounts are found outside the membrane.

Results reveal that amide-to-ester substitution in the gramicidin channel protein removes the hydrogen bond involved in the intramolecular stabilization between Gly and Val of N- and C-termini, respectively. Data further show that the substitution alters the charge distribution leading to intermolecular hydrogen bond disruption and subunit association and dissociation.

Research has been conducted on the lipid and membrane-spanning peptide interactions. The effect of the tryptophan-anchored hydrophobic peptides on the 1,2-dielaidoylphosphatidylethanolamine despersion phase behavior has been investigated.

The D-Leu(super 10) residue creates the optimal orientation of Trp(super 9) in gramicidin channels in lipid bilayers. Gramicidin A has four Trp residues, which are essential for channel assembly and stability and ion transport.

H nuclear magnetic resonance and erythrocyte sedimentation rate techniques were used to investigate the possible effects of a hydrophobic mismatch between proteins/lipids on membrane structure and function. The study focused on the possible shortening or stretching of the lipid acyl chains in bilayer systems that face a hydrophobic mismatch induced by transmembrane WALP or related uncharged alpha-helical peptides. Gramicidin was included for the purpose of comparison. Results show that the peptide hydrophobic length influences bilayer thickness in a systematic way to reduce mismatch.

Gramicidin A (gA) was labelled with ethanolamine-d4 and palmitoylated within hydrated dimyristoylphosphatidylcholine (DMPC) bilayers to assess acylation activities. Ethanolamine was found mobile before acylation, but became inert after palmitoylation. Possible structural conformations in palymitoylation may have the ethanolamine group rotating toward the outside of the gramicidin A cylinder upon acylation, with the acyl chain passing between the side chains of Trp9 and Leu10.

The effect of hydrophobic mismatch on the lipid structure of diacylphosphatidylcholines with varying length and unsaturation was investigated by circular dichroism and nuclear magnetic resonance experiments. The results showed that at high peptide/lipid ratios, the peptides induce nonbilayer structures in phosphatidylcholine, depending on the extent of mismatch. They also indicated that the amino acid tryptophan influences the formation of the lipid bilayer. The molecular model of induced membranes is presented.

Acylation is a subtle process for regulating interactions between lipids and proteins and for mediating the functions and stability of membrane proteins. By attaching palmitic acid to gramicidin A (gA), a study has been conducted to determine the effect of acylation on the backbone and the orientation and dynamics of the side chains of Val7, Trp9, Leu10 and Trp11, using 2-dimensional 1H-NMR spectroscopy. The covalently attached acyl chain initiates site-specific alterations in the side-chain orientation for Leu10 and Trp9 but not for Val7, Trp11 or the backbone. One beta-hydrogen of Leu10 of gA is protected by an adjacent aromatic ring, but this shielding does not occur after acylation.

Gramicidin dispersions show that gA channel development depends on the acyl chain length of diacylphosphatidylcholines, used to analyze the conformation of gA. gA disassociation fragments increase with increase in acyl chain length, and gA channel formation depends on gA and lipid concentration, solvent-history and temperature. The dioctanoyl-PC aggregate size is increased by the gA.

The structural and functional effects of alanine-to-serine substitutions in bilayer-spanning gramicidin channels are examined in order to analyze the consequences of burying polar, hydrogen-bonding hydroxyl groups within the hydrocarbon core of lipid bilayer membranes. The studies have shown that when compared to the corresponding Ala-containing reference channels, the polar Ser residues have decreased the analoguesE channel-forming potency, indicating a substantial energetic penalty for burying the polar Ser side chain in the bilayer hydrophobic core.

Both solid-state NMR experiments and coarse-grained molecular dynamics (CG-MD) simulations have shown that peptide sequence isomers having Arg in position 12 or position 14 has displayed markedly different behaviors. The presence of R14 has induced bilayer thinning and peptide displacement to 'lift' the charged guanidinium toward the bilayer surface.

Acetyl-GWWLALALAP10ALALALWWA-ethanolamide and included pairs of deuterated alanines was synthesized to measure the extent of helix kinking caused by a single proline within the isolated transmembrane (TM) helical domain of WALP19. The results provide insight into the influence of the central proline on the geometry and the dynamics of the membrane-spanning peptide and the functional role of proline in several families of membrane proteins.

The orientation and motion of the tryptophan interfacial anchors of integrated proteins in various membrane-spanning peptides are being studied. The analysis reveals that the tryptophans near the C-terminal end of the peptide are very different from those near the N-terminus.