Fluorescence Quenching and Fluorescence Resonance Energy Transfer Studies in the Recombinant N-domain from the Plasma Membrane H(+)-ATPase, Pma1

Abstract

Upon substrate binding the isolated plasma membrane H(+)-ATPase (Pma1) from Kluyveromyces lactis displays large changes in fluorescence intensity (Sampedro et al, 2007 Biochemistry 46:5616-5622). The nucleotide binding domain (N-domain) contains one Trp505 residue, that seems to be responsible for the variations in intrinsic fluorescence. The N-domain was cloned and the protein expressed in E. coli. The purified N-domain displayed nucleotide-dependent (ATP and ADP) quenching of fluorescence similar to that observed in the whole Pma1. The dissociation constants (Kd) for ATP and ADP were 100 and 110 uM respectively. Fluorescence resonance energy transfer (FRET) studies were also performed by using mantATP; a fluorescent ATP analog (Ex. 337 nm, Em. 423nm). The absorbance spectra of mantATP overlaps the fluorescence spectra of the N-domain, and thus FRET was observed by exciting at 280 nm. FRET efficiency was 100% indicating a close proximity between Trp505 and the nucleotide. Therefore, in this domain there is a Trp residue located near the substrate binding site which is of high value to determine Kds and molecular distances using fluorescence.