Molecular Dynamics of Mitochondrial Proton Conduction

Pringle, Michael

Abstract

Proton conduction through the H+ ATPase (F1-F0) is of crucial importance in mitochondrial energy transduction. The overall strategy of this proposal is to examine the structure, function, and molecular dynamics of components of the enzyme complex in defined lipid vesicles and to characterise the unit conductance of the proton channel in Fo by incorporating the channel into planar bilayers. The proposal seeks to identify the role of intersubunit and protein/lipid interactions in modulating the conduction of protons thrugh mitochondrial H+ ATPase. Specically, proteoliposomes will be prepared from our highly purified F1-F0 and F0 preparations and defined lipids. The butanol-extracted DCCD-binding protein will also be incorporated into liposomes. All these preparations will be made with and without coupling factor B (FB), a component of F0 required for ATP-Pi exchange. Lipid and protein analyses will identify the membrane composition of recombinants, while freeze-fracture electron microscopy will be used to study the lateral distribution of components and their aggregation state. Functional assays will involve measurements of oligomycin-sensitive ATPase and ATP-Pi exchange activities as well as ATP-driven proton conduction and passive proton flux through the proton channel (in liposomes and planar bilayers). To investigate whether the dynamic properties of the complexes correlate with their function, lipid and protein mobilities in the membrane vesicles will be measured by EPR (conventional and saturation transfer) and phosphorescence anisotropy. Temperature will be used as a probe of protein/lipid interactions in both the functional and dynamic properties of the recombinants. It is expected that the combination of biochemical and spectrosocpic techniques proposed here will further our understanding of mitochondrial proton translocation.