The Structural and Functional Role of the y-e Rotor in Escherichia Coli F0F1 ATP Synthase

Lin, Shin-Kai, Department of Molecular Physiology and Biological Physics, University of Virginia
Nakamoto, Robert, Department of Molecular Physiology and Biological Physics, University of Virginia
Cafiso, David, Department of Chemistry, University of Virginia

The --c subunits of the Escherichia coli F O F 1 ATP synthase make up the rotor assembly, which couples proton transport to ATP catalysis. Previous EPR and functional studies from our laboratory suggest that interactions in the - and -c interfaces play important roles in efficient coupling. To further investigate the structure and dynamics of the interface, we have used the site-directed spin labeling strategy of EPR spectroscopy to define the structure of  subunit in the - -c subunit interface. EPR spectra of isolated  subunits with spin labels at single-cysteine substitutions from E29C to A44C show an alternating mobility indicating a -strand secondary structure. When bound to F 1 complex, spectra of spin-labeled P40C-T43C show large decreases of mobility, which are likely due to tertiary contacts with the  subunit. The L42C mutant subunit binds to the F 1 complex with lower affinity indicating that the hydrophobic interaction contributes significant amount of binding energy in - interface. The EPR spectra of spin labeled 29, 33, 35, 36, 37 and 39 subunit-F 1 complexes demonstrate large mobility decreases when F 1 is reconstituted with the membranous F O sector, suggesting that these residues directly interact with subunit c. Single-cysteine mutations introduced in the -c interface do not disrupt energy coupling between F O and F 1 , implying that the -c interface is the main channel for the energy coupling mechanism and one role of  is to strengthen the -c interaction and ii coordinate conformations of  and c subunits for optimal coupling efficiency. Deuterium-Hydrogen exchange and X-ray footprinting methods were used to explore the solvent-accessibility changes of the rotor and rotor-stator interface regions in the presence and absence of ATP. However, due to the problem of deuterium-back-exchange, solvent accessibility map of the - rotor could not be established based on the deuterium-hydrogen exchange method. From X-ray footprinting analysis, the nucleotide-dependent changes of oxidative rates of Met178 and Met379 suggest the possible important contacts between  and  3  3 hexamer during rotation. Decreases of oxidative rates of Met15, Met49, and Phe61 in the presence of ATP provide evidence for the nucleotidedependent movement of C-terminal helices of  subunit.

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PHD (Doctor of Philosophy)
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