Strategies for elucidation of the structure and function of the large membrane protein complex, FoF1-ATP synthase, by nuclear magnetic resonance.

ABSTRACT

Nuclear magnetic resonance (NMR) investigation of large membrane proteins requires well-focused questions and critical techniques. Here, research strategies for FoF1-ATP synthase, a membrane-embedded molecular motor, are reviewed, focusing on the ß-subunit of F1-ATPase and c-subunit ring of the enzyme. Segmental isotope-labeling provided 89% assignment of the main chain NMR signals of thermophilic Bacillus (T)F1ß-monomer. Upon nucleotide binding to Lys164, Asp252 was shown to switch its hydrogen-bonding partner from Lys164 to Thr165, inducing an open-to-closed bend motion of TF1ß-subunit. This drives the rotational catalysis. The c-ring structure determined by solid-state NMR showed that cGlu56 and cAsn23 of the active site took a hydrogen-bonded closed conformation in membranes. In 505 kDa TFoF1, the specifically isotope-labeled cGlu56 and cAsn23 provided well-resolved NMR signals, which revealed that 87% of the residue pairs took a deprotonated open conformation at the Foa-c subunit interface, whereas they were in the closed conformation in the lipid-enclosed region.