Structural mapping of nucleotide binding sites on chloroplast coupling factor

Abstract

Fluorescence resonance energy transfer was used to measure the distances between 3 nucleotide binding sites on solubilized chloroplast coupling factor from spinach and between each nucleotide site and 2 tyrosine residues which are important for catalytic activity. The nucleotide energy donor was 1,N6-ethenoadenosine di- or triphosphate and the nucleotide energy acceptor was 2''(3'')-(trinitrophenyl) adenosine diphosphate. The tyrosine residues were specifically labeled with 7-chloro-4-nitro-2,1,3-benzoxadiazole, which served as an energy acceptor. Apparently, the 3 nucleotide binding sites form a triangle with sides of 44, 48 and 36 .ANG.. (The assumption was made in calculating these distances that the energy donor and acceptor rotate rapidly relative to the fluorescence lifetime.) Two of the nucleotide sites are approximately equidistant from each of the 2 tyrosines: one of the nucleotide sites is .apprx. 37 .ANG. and the other .apprx. 41 .ANG. from each tyrosine. The 3rd nucleotide site is > 41 .ANG. from one of the tyrosines and .gtoreq. 41 .ANG. from the other tyrosine.