) The relationship between the chemistry of ATP hydrolysis and the mechanical events that result in rotation of the gamma subunit of the F1-ATPase will be examined. Catalytic function of F1 derives from the asymmetry of the catalytic sites that, in turn, depends on the gamma subunit and the Mg2+ cofactor. The driving force for gamma rotation is believed to result from the initial binding energy of the Mg2+-ATP complex and from the release of phosphate which is a Mg2+ ligand. The ability of the gamma subunit of F1 to rotate will be measured using a fluorescent microsphere attached to the gamma subunit as recorded using a CCD camera. The torque generated during gamma rotation as well as the dwell time between rotations will be as assessed with F1 that contains site-directed mutants or other treatments at locations that may affect the coupling between hydrolysis and gamma rotation. Three loci are targeted for investigation that include: (a) Switch 3, the gamma subunit C-terminus and the beta subunit greasy bearing which is close to the site of Mg2+ binding and phosphate release; (b) Switch 2, the interface between the gamma subunit and the beta subunit DELSEED sequence; and (c) Switch 1, where hydrogen bonds form between the gamma subunit and the betaE subunit catch loop. Experiments will examine the possibility that Switch 1 is part of an escapement mechanism that only allows gamma rotation when the catalytic sites are filled with metal-nucleotide complex. Experiments are also designed to identify changes in metal ligands at the catalytic sites that are specifically associated with conformational changes linked to gamma rotation.
Showing the most recent 10 out of 19 publications
