Structure and Function Studies of Energy and Signal Transducing Proteins by Solid-State NMR

Abstract

A series of pulse sequences for the sequential assignment of proteins were developed and applied to mastoparan-X(MPx) and H+-ATP synthase subunit c (Fo) in membrane systems. MPx is a model peptide of a loop of G-protein coupled receptor that activates G-protein. A backbone structure of MPx tightly bound to membranes and the mode of MPx/membrane interaction were determined at atomic resolution. The latter suggested a role of membranes in the efficient G-protein activation by membrane-bound MPx. The secondary structure of Fo from E. coli (EFoc) in the solid state was estimated by TALOS on the basis of chemical shifts. It was mainly composed of two α-helical regions with a loop in the middle (residues 41–45) as in the case of the solution structure. Solid-state 2H NMR analysis of the EFoc/DMPC-d54 system revealed that in the liquid-crystalline state, the subunit c interacts with the lipids nearly in the same manner as the fluid lipids interact among themselves. This is achieved by the hydrophobic matching in the thickness and the soft interaction between the protein and hydrocarbon chains of lipids. In contrast, the hydrophobic matching is broken in the gel state. Keywords: membrane protein; mastoparan-X; G-protein; G-protein-coupled receptor; F1Fo-ATP synthase; subunit c-ring; lipid bilayer; hydrophobic matching; viscoelasticity