Protein Rotational Dynamics in Aligned Lipid Membranes Probed by Anisotropic T_1ρ NMR Relaxation

A membrane-bound form of Pf1 coat protein reconstituted in magnetically aligned DMPC/DHPC bicelles was used as a molecular probe to quantify for the viscosity of the lipid membrane interior by measuring the uniaxial rotational diffusion coefficient of the protein. Orientationally dependent ¹⁵N NMR relaxation times in the rotating frame, or T_1ρ, were determined by fitting individually the decay of the resolved NMR peaks corresponding to the transmembrane helix of Pf1 coat protein as a function of the spin-lock time incorporated into the 2D SAMPI4 pulse sequence. The T_1ρ relaxation mechanism was modeled by uniaxial rotational diffusion on a cone, which yields a linear correlation with respect to the bond factor sin⁴θ_B, where θ_B is the angle that the NH bond forms with respect to the axis of rotation. Importantly, the bond factors can be independently measured from the dipolar couplings in the separated local-field SAMPI4 spectra. From this dependence, the value of the diffusion coefficient D_|| = 8.0 × 10⁵ s^-1 was inferred from linear regression of the experimental T_1ρ data even without any spectroscopic assignment. Alternatively, a close value of D_|| = 7.7 × 10⁵ s^-1 was obtained by fitting the T_1ρ relaxation data for the assigned NMR peaks of the transmembrane domain of Pf1 to a wavelike pattern as a function of residue number. The method illustrates the use of single-helix transmembrane peptides as molecular probes to assess the dynamic parameters of biological membranes by NMR relaxation in oriented lipid bilayers.