ATP BINDING SITE PHOTOAFFINITY PROBES FOR F1-ATPASE

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The long term goal of this research is to clarify the mechanism of the multisubunit F1-ATPase, one of the most important yet structurally complex enzymes, ubiquitously responsible for creation most of the ATP of normal and diseased cells. It consists of two parts, one of which is membrane bound and serves as a transmembrane proton channel (F0); the other part performs the catalytic synthesis of ATP from ADP + Pi(F1). The F1 requires for catalysis cooperative participation of at least 2 catalytic nucleotide binding sites on different (beta) subunits. Newly designed fluorescent photoaffinity nucleotide analog probes (BzAF and BzAE, the latter possessing heavy iodine atoms), which covalently crosslink to F1 only at its 3 catalytic sites, indicate that when these sites are sequentially occupied, a pre-catalytic conformational change in F1 occurs. This causes a coordinated change in the affinity of each site for the bound substrate ligands, a process consonant with the currently accepted "binding change/alternating site" mechanism. Together with our previously designed and successfully employed set of benzophenone-derivatized nucleotide photoaffinity labels (BzAT(D)p), the questions to be addressed for clarification are: 1) What differences exist in the specific Beta subunit peptide domains and amino acid residues that bind nucleotide when one vs. more-than-one mol of substrate analog ligand is covalently linked to a catalytic site per mol enzyme? 2) Does the existence of a transmembrane proton gradient contribute to a conformational change in F1? 3) What is the distance between the cooperatively interacting catalytic nucleotide binding sites on the F1 holoenzyme? 4) What is the specific topological location of the catalytic (beta subunit) nucleotide site(s) within the F1 multisubunit holoenzyme as deduced via X-ray crystallography? 5) What ate the conformational adjustments required for catalysis by other multinucleotide-utilizing enzymes, such as muscle adenylate kinase (ADK) as disclosed via our new fluorescent, nucleotide analog photoaffinity probes? Experimental methods to solve these problems shall include: 1) Crosslinking of various species of F1 (bacterial as well as mitochondrial) to controlled labelling stoichiometries with our arsenal of Bz-photoaffinity probes (BzAT(D,M)P,BzAF and BzAE;2) High resolution protein sequencing of F1 covalently labelled with on vs. more-than-one mol of Bz probe; 3) Steady state fluorescence studies of proton gradient-induced, vs. sequential binding site occupancy-induced, conformational changes in F1 in reconsituted membrane vesicle systems; 4) Use of the florescent BzAF and BzAE (or AdN-Tb3+) probes as spectroscopic pairs with bi-labelled F1 to characterize interactions and measurements of distances between catalytic sites, via Forster energy transfer; 5) Use of the tetraiodinated BzAE as an intrinsic topological marker of nucleotide binding in the x-ray structure of the BzAE labelled F1 crystal.