Orientation and dynamics of synthetic transbilayer polypeptides containing GpATM dimerization motifs.

Deuterium NMR spectroscopy was used to study how the positioning of a dimerization motif within a transbilayer polypeptide influences its orientation and dynamics in bilayers. Three polypeptide variants comprising glycophorin A transmembrane (GpATM) dimerization motifs incorporated into lysine-terminated poly-leucine-alanine helices were mixed into 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine multilamellar vesicles. The variants differed in orientation of the motif segment around the helix axis with respect to the peptide ends. Polypeptides were labeled with methyl-deuterated alanines at positions that were identically situated relative to the peptide ends (Ala-20 and Ala-22) and at two positions within the motif. An analysis of quadrupole splittings revealed similar tilts and orientations of the peptide ends for all three variants, suggesting that average orientations were dominated by interactions at the bilayer surface. For one variant, however, fast orientational fluctuations about the helix axis were significantly smaller. This may indicate some perturbation of peptide dynamics and conformation by interactions that are sensitive to the motif orientation relative to the peptide ends. For the variant that displayed distinct dynamics, one orientation consistent with observed splittings corresponded to the motif being situated such that its two glycines were particularly accessible to adjacent peptides.

Influence of DPH on the structure and dynamics of a DPPC bilayer.

We have conducted extensive molecular dynamics (MD) simulations together with differential scanning calorimetry (DSC) and nuclear magnetic resonance (NMR) experiments to quantify the influence of free 1,6-diphenyl-1,3,5-hexatriene (DPH) fluorescent probes on the structure and dynamics of a dipalmitoylphosphatidylcholine bilayer. Atomistic MD simulations show that in the membrane-water interface the influence of DPH is minor, whereas in the acyl-chain region DPH gives rise to major perturbations. In the latter case, DPH is found to influence a wide range of membrane properties, such as the packing and ordering of hydrocarbon tails and the lateral diffusion of lipid molecules. The effects are prominent but of local nature, i.e., the changes observed in the properties of lipid molecules are significant in the vicinity of DPH, but reduce rapidly as the distance from the probe increases. Long-range perturbations due to DPH are hence not expected. Detailed DSC and (2)H NMR measurements support this view. DSC shows only subtle perturbation to the cooperative behavior of the membrane system in the presence of DPH, and (2)H NMR shows that DPH gives rise to a slight increase in the lipid chain order, in agreement with MD simulations. Potential effects of other probes such as pyrene are briefly discussed.