PROJECT SUMMARY
F1Fo ATP synthase is a fundamental energy conversion complex found in all known cells. This complex uses an
electrochemical gradient of ions to drive a rotary mechanism that results in the synthesis of adenosine
triphosphate (ATP), the universal chemical energy currency of life. In E. coli, the proton-driven Fo motor,
embedded in the cell membrane, consists of a decameric subunit c rotor ring in contact with subunit a of the
stator. Protons move through Fo via two offset aqueous half channels. Protons enter through subunit a from
the periplasm (P-side) to the c-ring. After c-ring rotation, protons exit to the cytoplasm (N-side) by a half channel
formed at the a-c interface. Clusters of residues along the N-side channel have been implicated in proton
translocation and a possible gating mechanism, but the roles of these residues are unclear. This project seeks
to engage undergraduate student researchers in a combined biochemical and biophysical approach to
characterize interactions between the rotor and stator and establish their role(s) in proton transport. Aim 1 will
systematically examine mutations and chemical modifications of several key residues on the N-side of the a-c
interface with a battery of in vitro biochemical assays to determine the role of each residue in proton-driven
ATP synthesis and ATP-driven proton pumping. Aim 2 will use site-directed spin labeling and electron
paramagnetic resonance (EPR) spectroscopy to probe the structure and structural dynamics of the loop regions
of subunit a that interact with the c-ring. Patterns of spin label mobility, accessibility, and proximity will reveal
whether interacting cytoplasmic loops at the a-c interface undergo conformational changes during ATP
synthesis or hydrolysis. Together, the structural and functional data will contribute to understanding the
molecular mechanism of proton transport in this important biomolecular machine and inform future drug
development.
