Conformational Changes of the Nucleotide Site of the Plasma Membrane Ca2+-ATPase Probed by Fluorescence Quenching

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Abstract
Fluorescence quenching by the water-soluble ions I- and Cs+ was used to probe solvent accessibility and polarity of the nucleotide/fluorescein isothiocyanate binding pocket of the purified soluble Ca2+-ATPase from plasma membranes. The E1·Ca·CaM conformer was the least accessible state studied, presenting the lowest suppression constant (Kq) for both I- (Kq = 6.7 M-1) and Cs+ (Kq = 0.7 M-1). Accessibility to I- was similar for the E2·VO4 and E1·Ca states (Kq = 7.13 and 7.5 M-1, respectively), whereas E2 was slightly more accessible (Kq = 9.1 M-1). The phosphorylated state E2-P presented the highest accessibility, with a Kq of 16.5 M-1, very near the Kq of 20.3 M-1 for free FITC. I- was unequivocally a better fluorescence quencher, being usually nearly 3-fold as efficient as Cs+, as indicated by the Kq(I-)/Kq(Cs+) ratio (Rq). The advent of a positive charge cluster on the nucleotide/fluorescein binding pocket in different states was suggested by the increase in Rq, which reached a value as high as 9.5 for the E1·Ca·CaM conformer. These results indicate (i) a very high water accessibility of the nucleotide/fluorescein pocket for E2-P that (ii) is more restricted on the free E2 state and (iii) becomes rather lower for the E1·Ca states. Additionally, a positive charge effect of amino acids on the nucleotide site, possibly related to ATP binding and phosphoryl transfer, appears in these E1·Ca states, being absent in the phosphorylated and nonphosphorylated E2 states.

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