Supported Tubulated Bilayers: A Novel System for Evaluating Protein-Mediated Membrane Remodeling

Abstract

Transmembrane domains will sometimes contain conserved ionizable residues which are essential for protein function and regulation. This work aims to examine the effects of single Arg(R) residues within a highly dynamic transmembrane peptide helix. We have modified the dynamic transmembrane GW 4,20 ALP23 (acetyl-GGAW 4 (AL) 7 AW 20 AGA-[ethanol] amide) peptide to incorporate an Arg residue near the center of the peptide at position 12 or 14. Peptide orientation and dynamics were analyzed by means of solid-state NMR spectroscopy to monitor specific 2 H-labeled Ala residues. GW 4,20 ALP23 adopts a tilted orientation within lipid bilayers and exhibits moderate to high dynamic averaging of NMR observables, such as the 2 H quadrupolar splittings or 15 N-1 H dipolar couplings, due to competition between the interfacial Trp(W) residues on opposing helical faces. Here we examine how such extensive peptide dynamics are impacted by the introduction of central Arg residues. R14 holds significant influence over the peptide's behavior and restricts the peptide to low dynamics and a tilt that decreases as lipid thickness increases, all at the cost of partial C-terminal helix unwinding. Alternatively, R12 causes the host peptide to adopt an interfacial surface-bound orientation in DOPC bilayers. Interestingly, multistate behavior is exhibited by a single residue, Ala-9. Furthermore, numerous N-terminal quadrupolar splittings generated by 2 H-labeled residues do not fit to the same quadrupolar wave plot as the remaining C-terminal residues. Therefore, both results potentially implicate distortion of the R12 helix. The partial helix unwinding/distortion behavior observed in both the R12 and R14 peptides could be due to the competition of the terminal Trp residues attempting to compensate for the Arg's dominance over the peptide's orientation. We thus surmise that while the arginine residues are prominent factors controlling the helix dynamics, the influence of the competing tryptophan residues cannot be ignored.