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

Solid-State NMR Ensemble Dynamics as a Mediator between Experiment and Simulation

Solid-state NMR (SSNMR) is a powerful technique to describe the orientations of membrane proteins and peptides in their native membrane bilayer environments. The deuterium (2H) quadrupolar splitting (DQS), one of the SSNMR observables, has been used to characterize the orientations of various single-pass transmembrane (TM) helices using a semistatic rigid-body model such as the geometric analysis of labeled alanine (GALA) method. However, dynamic information of these TM helices, which could be related to important biological function, can be missing or misinterpreted with the semistatic model. We have investigated the orientation of WALP23 in an implicit membrane of dimyristoylglycerophosphocholine by determining an ensemble of structures using multiple conformer models with a DQS restraint potential. When a single conformer is used, the resulting helix orientation (tilt angle (τ) of 5.6 ± 3.2° and rotation angle (ρ) of 141.8 ± 40.6°) is similar to that determined by the GALA method. However, as the number of conformers is increased, the tilt angles of WALP23 ensemble structures become larger (26.9 ± 6.7°), which agrees well with previous molecular dynamics simulation results. In addition, the ensemble structure distribution shows excellent agreement with the two-dimensional free energy surface as a function of WALP23's τ and ρ. These results demonstrate that SSNMR ensemble dynamics provides a means to extract orientational and dynamic information of TM helices from their SSNMR observables and to explain the discrepancy between molecular dynamics simulation and GALA-based interpretation of DQS data

Charged or Aromatic Anchor Residue Dependence of Transmembrane Peptide Tilt*

The membrane-spanning segments of integral membrane proteins often are flanked by aromatic or charged amino acid residues, which may “anchor” the transmembrane orientation. Single spanning transmembrane peptides such as those of the WALP family, acetyl-GWW(LA)nLWWA-amide, furthermore adopt a moderate average tilt within lipid bilayer membranes. To understand the anchor residue dependence of the tilt, we introduce Leu-Ala “spacers” between paired anchors and in some cases replace the outer tryptophans. The resulting peptides, acetyl-GX2ALW(LA)6LWLAX22A-amide, have Trp, Lys, Arg, or Gly in the two X positions. The apparent average orientations of the core helical sequences were determined in oriented phosphatidylcholine bilayer membranes of varying thickness using solid-state 2H NMR spectroscopy. When X is Lys, Arg, or Gly, the direction of the tilt is essentially constant in different lipids and presumably is dictated by the tryptophans (Trp5 and Trp19) that flank the inner helical core. The Leu-Ala spacers are no longer helical. The magnitude of the apparent helix tilt furthermore scales nicely with the bilayer thickness except when X is Trp. When X is Trp, the direction of tilt is less well defined in each phosphatidylcholine bilayer and varies up to 70° among 1,2-dioleoyl-sn-glycero-3-phosphocholine, 1,2-dimyristoyl-sn-glycero-3-phosphocholine, and 1,2-dilauroyl-sn-glycero-3-phosphocholine bilayer membranes. Indeed, the X = Trp case parallels earlier observations in which WALP family peptides having multiple Trp anchors show little dependence of the apparent tilt magnitude on bilayer thickness. The results shed new light on the interactions of arginine, lysine, tryptophan, and even glycine at lipid bilayer membrane interfaces

Exploring Peptide-Membrane Interactions with Coarse-Grained MD Simulations

The interaction of α-helical peptides with lipid bilayers is central to our understanding of the physicochemical principles of biological membrane organization and stability. Mutations that alter the position or orientation of an α-helix within a membrane, or that change the probability that the α-helix will insert into the membrane, can alter a range of membrane protein functions. We describe a comparative coarse-grained molecular dynamics simulation methodology, based on self-assembly of a lipid bilayer in the presence of an α-helical peptide, which allows us to model membrane transmembrane helix insertion. We validate this methodology against available experimental data for synthetic model peptides (WALP23 and LS3). Simulation-based estimates of apparent free energies of insertion into a bilayer of cystic fibrosis transmembrane regulator-derived helices correlate well with published data for translocon-mediated insertion. Comparison of values of the apparent free energy of insertion from self-assembly simulations with those from coarse-grained molecular dynamics potentials of mean force for model peptides, and with translocon-mediated insertion of cystic fibrosis transmembrane regulator-derived peptides suggests a nonequilibrium model of helix insertion into bilayers

Probing the lipid-protein interface using model transmembrane peptides with a covalently linked acyl chain

The aim of this study was to gain insight into how interactions between proteins and lipids in membranes are sensed at the protein-lipid interface. As a probe to analyze this interface, we used deuterium-labeled acyl chains that were covalently linked to a model transmembrane peptide. First, a perdeuterated palmitoyl chain was coupled to the Trp-flanked peptide WALP23 (Ac-CGWW(LA)<sub>8</sub>LWWA-NH<sub>2</sub>), and the deuterium NMR spectrum was analyzed in di-C18:1-phosphatidylcholine (PC) bilayers. We found that the chain order of this peptide-linked chain is rather similar to that of a noncovalently coupled perdeuterated palmitoyl chain, except that it exhibits a slightly lower order. Similar results were obtained when site-specific deuterium labels were used and when the palmitoyl chain was attached to the more-hydrophobic model peptide WLP23 (Ac-CGWWL<sub>17</sub>WWA-NH<sub>2</sub>) or to the Lys-flanked peptide KALP23 (Ac-CGKK(LA)<sub>8</sub>LKKA-NH<sub>2</sub>). The experiments showed that the order of both the peptide-linked chains and the noncovalently coupled palmitoyl chains in the phospholipid bilayer increases in the order KALP23 < WALP23 < WLP23. Furthermore, changes in the bulk lipid bilayer thickness caused by varying the lipid composition from di-C14:1-PC to di-C18:1-PC or by including cholesterol were sensed rather similarly by the covalently coupled chain and the noncovalently coupled palmitoyl chains. The results indicate that the properties of lipids adjacent to transmembrane peptides mostly reflect the properties of the surrounding lipid bilayer, and hence that (at least for the single-span model peptides used in this study) annular lipids do not play a highly specific role in protein-lipid interactions