Biophysical studies of the b subunit of the Escherichia coli ATP synthase

The ATP synthase produces most of the ATP for both eukaryotic cells and aerobic bacteria and is critical for their proper functioning and survival. The mechanism of ATP synthesis involves the interaction of the integral membrane and the soluble sectors of this enzyme. Each of these can operate independently as a rotary motor protein. The coupling of these two motors in the production of ATP has become an area of intense study. The structure of the central rotor has been solved at high resolution and is well understood. The membrane and soluble portions of the stationary section of this complex are linked by a stalk on the periphery of the complex formed by the two copies of the b subunit and the δ subunit. The stalk functions as a link that holds the soluble sector to the membrane sector and may have an additional role in the rotary catalytic mechanism including the ability to store elastic energy. The structure of the soluble region of the b subunit is not known but it has been shown to stretch from the membrane surface to the upper portion of the soluble sector a distance of up to 140 Å. The physical properties of the 132 amino acid soluble domain of b has been the object of study in this project with a goal of gaining detailed knowledge of its structure that is relevant to the proposed roles in the catalytic mechanism of the complex. Hydrodynamic studies of a series of truncations of b resulted in the delineation of domains in the soluble region responsible for dimer formation between the two copies of b and of the C-terminal region that binds the δ. SAXS and NMR studies of the dimerization domain showed that is an extended structure consistent with a pair of extended helices. CD and thermodynamic analysis indicate a weak coiled coil dimer interface. Other work identified residues involved in the interface between the C-termini of b and δ. This work has allowed the development a model of the b subunit that has implications for its role in the ATP synthase complex.