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• W2003040695 endingPage "39082" @default.
• W2003040695 startingPage "39070" @default.
• W2003040695 abstract "We have used fluorescence spectroscopy, molecular modeling, and limited proteolysis to examine structural dynamics of the sarcoplasmic reticulum Ca-ATPase (SERCA). The Ca-ATPase in sarcoplasmic reticulum vesicles from fast twitch muscle (SERCA1a isoform) was selectively labeled with fluorescein isothiocyanate (FITC), a probe that specifically reacts with Lys-515 in the nucleotide-binding site. Conformation-specific proteolysis demonstrated that FITC labeling does not induce closure of the cytoplasmic headpiece, thereby assigning FITC-SERCA as a nucleotide-free enzyme. We used enzyme reverse mode to synthesize FITC monophosphate (FMP) on SERCA, producing a phosphorylated pseudosubstrate tethered to the nucleotide-binding site of a Ca(2+)-free enzyme (E2 state to prevent FMP hydrolysis). Conformation-specific proteolysis demonstrated that FMP formation induces SERCA headpiece closure similar to ATP binding, presumably due to the high energy phosphoryl group on the fluorescent probe (ATP·E2 analog). Subnanosecond-resolved detection of fluorescence lifetime, anisotropy, and quenching was used to characterize FMP-SERCA (ATP·E2 state) versus FITC-SERCA in Ca(2+)-free, Ca(2+)-bound, and actively cycling phosphoenzyme states (E2, E1, and EP). Time-resolved spectroscopy revealed that FMP-SERCA exhibits increased probe dynamics but decreased probe accessibility compared with FITC-SERCA, indicating that ATP exhibits enhanced dynamics within a closed cytoplasmic headpiece. Molecular modeling was used to calculate the solvent-accessible surface area of FITC and FMP bound to SERCA crystal structures, revealing a positive correlation of solvent-accessible surface area with quenching but not anisotropy. Thus, headpiece closure is coupled to substrate binding but not active site dynamics. We propose that dynamics in the nucleotide-binding site of SERCA is important for Ca(2+) binding (distal allostery) and phosphoenzyme formation (direct activation)." @default.
• W2003040695 created "2016-06-24" @default.
• W2003040695 creator A5016536937 @default.
• W2003040695 creator A5063616345 @default.
• W2003040695 creator A5064723424 @default.
• W2003040695 creator A5064803348 @default.
• W2003040695 creator A5074917182 @default.
• W2003040695 date "2012-11-01" @default.
• W2003040695 modified "2023-10-03" @default.
• W2003040695 title "Nucleotide Activation of the Ca-ATPase" @default.
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