Dynamic and Contrasting Information by Oriented-Sample Solid-State NMR Spectroscopy of Membrane Proteins

Abstract

Oriented-sample NMR (OS NMR) has emerged as a powerful technique for structure determination of membrane proteins in their native-like lipid environment. We have developed a model relating OS NMR lineshapes to uniaxial ordering (mosaic spread) and rotational diffusion of the protein within the membrane. The model is exemplified by 15N NMR spectra of Pf1 coat protein in both magnetically aligned phage and reconstituted in oriented bicelles. In the case of Pf1 phage, the lineshapes are dominated by static uniaxial disorder; whereas fast rotational diffusion of the protein is responsible for the motional line narrowing in perpendicularly oriented bicelles. From the analysis of 15N NMR linewidths, rotational diffusion coefficient can be estimated. Since the value of the diffusion coefficient is ultimately related to the overall protein size, information about oligomerization states is potentially obtainable. Second, the use of various membrane-embedded radicals allows one to both dramatically speed up data acquisition, on the one hand, and obtain contrasting information for membrane-embedded proteins, on the other. While membrane-bound paramagnetic species drastically affect the T1Z relaxation times (at 2:1 molar ratio relative to the protein), the transverse T2 relaxation is only slightly affected. 5-DOXYL stearic acid, TEMPOL, and CAT-1 radicals exhibit different partitioning within the membrane, and result in differential paramagnetic effect on the spectral peaks arising from different residues of Pf1 protein in bicelles. This allows one to obtain contrasting information about the location of the residues relative to the membrane. As was shown by EPR, TEMPOL partitions itself equally in and out of the membrane, and almost uniformly affects all residues within the bilayer. By contrast, 5-DOXYL stearic acid affects mostly the residues below the interfacial region, while CAT-1 affects the residues located within the polar head groups.