On the combined analysis of ²H and ¹⁵N/ ¹H solid-state NMR data for determination of transmembrane peptide orientation and dynamics

The dynamics of membrane-spanning peptides have a strong affect on the solid-state NMR observables. We present a combined analysis of ²H-alanine quadrupolar splittings together with ¹⁵N/ ¹H dipolar couplings and ¹⁵N chemical shifts, using two models to treat the dynamics, for the systematic evaluation of transmembrane peptides based on the GWALP23 sequence (acetyl-GGALW(LA) ₆LWLAGA- amide). The results indicate that derivatives of GWALP23 incorporating diverse guest residues adopt a range of apparent tilt angles that span 5°-35° in lipid bilayer membranes. By comparing individual and combined analyses of specifically ²H- or ¹⁵N-labeled peptides incorporated in magnetically or mechanically aligned lipid bilayers, we examine the influence of data-set size/identity, and of explicitly modeled dynamics, on the deduced average orientations of the peptides. We conclude that peptides with small apparent tilt values (<∼10°) can be fitted by extensive families of solutions, which can be narrowed by incorporating additional ¹⁵N as well as ²H restraints. Conversely, peptides exhibiting larger tilt angles display more narrow distributions of tilt and rotation that can be fitted using smaller sets of experimental constraints or even with ²H or ¹⁵N data alone. Importantly, for peptides that tilt significantly more than 10° from the bilayer-normal, the contribution from rigid body dynamics can be approximated by a principal order parameter.