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• W2034247194 endingPage "5270" @default.
• W2034247194 startingPage "5261" @default.
• W2034247194 abstract "By analyzing, after expression in yeast and purification, the intrinsic fluorescence properties of point mutants of rabbit Ca2+-ATPase (SERCA1a) with alterations to amino acid residues in Ca2+-binding site I (E771), site II (E309), in both sites (D800), or in the nucleotide-binding domain (W552), we were able to follow the conformational changes associated with various steps in the ATPase catalytic cycle. Whereas Ca2+ binding to purified wild-type (WT) ATPase in the absence of ATP leads to the rise in Trp fluorescence expected for the so-called E2 → E1Ca2 transition, the Ca2+-induced fluorescence rise is dramatically reduced for the E309Q mutant. As this purified E309Q mutant retains the ability to bind Ca2+ at site I (but not at site II), we tentatively conclude that the protein reorganization induced by Ca2+ binding at site II makes the major contribution to the overall Trp fluorescence changes observed upon Ca2+ binding to both sites. Judging from the fluorescence response of W552F, similar to that of WT, these changes appear to be primarily due to membranous tryptophans, not to W552. The same holds for the fluorescence rise observed upon phosphorylation from Pi (the so-called E2 → E2P transition). As for WT ATPase, Mg2+ binding in the absence of Ca2+ affects the fluorescence of the E309Q mutant, suggesting that this Mg2+-dependent fluorescence rise does not reflect binding of Mg2+ to Ca2+ sites; instead, Mg2+ probably binds close to the catalytic site, or perhaps near transmembrane span M3, at a location recently revealed by Fe2+-catalyzed oxidative cleavage. Mutation of W552 hardly affects ATP-induced fluorescence changes in the absence of Ca2+, which are therefore mostly due to membranous Trp residues, demonstrating long-range communication between the nucleotide-binding domain and the membranous domain." @default.
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• W2034247194 date "2006-04-01" @default.
• W2034247194 modified "2023-10-10" @default.
• W2034247194 title "Conformational Changes in Sarcoplasmic Reticulum Ca²⁺-ATPase Mutants: Effect of Mutations either at Ca²⁺-Binding Site II or at Tryptophan 552 in the Cytosolic Domain" @default.
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• W2034247194 doi "https://doi.org/10.1021/bi0522091" @default.
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