Abstract

ATP synthase is a membrane embedded protein that is ubiquitous among all forms of life. ATP synthase is the primary site of ATP production, which makes this protein crucial for many biological processes. ATP synthase is divided into two coupled motors denoted F1 and Fo. F1 is water soluble and contains the active site where ATP synthesis and hydrolysis occur through conformational changes. Fo is the membrane-embedded motor composed of a c-10-ring and ab 2 subunits, that drives the conformational changes in F1 via a rotary mechanism in which protons flow down a potential gradient through two aqueous half channels in subunit a. Although there is considerable biochemical understanding of the mechanisms that occur in F1, the processes that facilitate proton translocation in Fo are still not well understood. Structures of subunit a determined by cryo-electron microscopy (cryo-EM) reveals inconsistences in conformations of this subunit when compared to previous crosslinking studies. This inconsistency suggests that multiple conformational states are taken on by subunit a which could provide insight to the mechanism of proton translocation. While detergent solubilization methods prove effective for purifying ATP synthase, they do not account for any protein-membrane interactions that may occur. We instead are developing a SMALPs (styrene-maleic acid lipid particle) technique to extract ATP synthase into lipid nano discs allowing the protein to be studied in its native lipid environment. In nanodiscs, protein dynamics can be probed by electron paramagnetic resonance (EPR) spectroscopy, and compared to that of soluble protein in order to determine more precisely the effect the membrane has on the protein mechanism. Small scale extractions have been performed with several different SMALPs to varying degrees of efficiency, but large-scale extractions still prove unsuccessful. Successful EPR data could help shed light on the involvement of lipids in the Fo mechanism of ATP synthase.

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