The long term goal of the project is to develop an understanding of the mechanism of energy conservation by the energy dependent F-0 F-1-ATPase of the Escherichia coli plasma membrane. The structure of the E. coli enzyme hears considerable homology to the mammalian mitochondrial ATPase. An understanding of how energy is conserved in these systems is of fundamental importance to human health and disease. Because it is clear that energy-dependent conformational changes in the ATPase are critical features of catalysis, the project will explore these conformational changes using endogenous tryptophan phosphorescence as a probe. Of central importance to the program is the E. coli mutant, recently developed in another laboratory, that is devoid of endogenous tryptophan. Using site-directed mutagenesis, single tryptophan residues will be inserted into selected positions of the molecule. Insertion of tryptophan into an alpha or beta subunit will introduce 3 residues per oligomer since a single gene codes for these subunits. The program will use phosphorescence to ask whether the microenvironments of the tryptophans are identical or different and whether the presence or absence of adenine nucleotides in nucleotide binding sites influences these microenvironments. The program will also ask, again using endogenous tryptophan phosphorescence as a probe, if the conformational changes said to accompany ADP binding as well as ATP binding and hydrolysis in recently reported X-ray diffraction studies of the mitochondrial enzyme, are kinetically competent. That is, are the structural changes at least as fast as catalysis?
