Membrane proteins embedded in liquid-like bilayers can undergo uniaxial rotational diffusion about the membrane normal. Measurements of the local bilayer viscosity can provide quantitative information about the environment surrounding membrane-embedded proteins. Using Pf1 coat protein reconstituted in aligned DMPC/DHPC bicelles as a molecular probe, its rotational diffusion coefficient was assessed by fitting the 15N T2 and T1ρ NMR relaxation rates. The relaxation rates were found to be highly anisotropic, which arise from the orientationally dependent heteronuclear dipolar couplings. A model for interpreting the anisotropic relaxation rates has been developed based on uniaxial diffusion on a cone. The model yields a linear correlation with respect to the bond factor sin4θB, where θB is the 1H-15N bond angle for each amide side. Importantly, the value of (3cos2θB -1) is directly measureable from the two-dimensional dipolar NMR spectrum. The diffusion coefficient D|| = 8.0×105 s-1 was determined from the simultaneous linear regression fitting of the experimental T1ρ data for each individual resolved NMR peak along the Pf1 backbone without any spectroscopic assignment. Moreover, the assigned T1ρ relaxation data for the helical domain of Pf1 can be fitted to a wave-like pattern having a periodicity of 3.6, which yields a value of D|| = 7.7×105 s-1. A reasonably close value (D|| = 5×105 s-1) has been independently obtained from fitting the T2 relaxation times. The experimentally obtained diffusion coefficients are consistent with simple estimates using the Stokes-Einstein equation if the protein is modeled as a rigid cylinder having the radius of 10 Å and local bilayer viscosity of 1 Poise.

Uniaxial diffusion of membrane proteins in aligned lipid bilayers probed by T2 and T1ρ NMR relaxation

ACS Spring 2022

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