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  • Title: Estimation of the distance change between cysteine-457 and the nucleotide binding site when sodium pump changes conformation from E1 to E2 by fluorescence energy transfer measurements.
    Author: Lin SH, Faller LD.
    Journal: Biochemistry; 1996 Jun 25; 35(25):8419-28. PubMed ID: 8679600.
    Abstract:
    The first indication of the size of a conformational change implicated in ion transport by sodium pump has been obtained by measuring the change in efficiency of fluorescence energy transfer between two specific locations on the alpha-subunit. The donor (5'-(iodoacetamido)fluorescein) attaches covalently to cysteine-457, and the acceptor (2'(or 3')-O-(trinitrophenyl)adenosine 5'-triphosphate) binds reversibly to the active site. The acceptor binds nearly 2 orders of magnitude tighter to the Na+ than to the K+ conformation of the enzyme and quenches donor fluorescence more efficiently in the Na+ than in the K+ conformation. The estimated distance between donor and acceptor, assuming random orientation of their emission and absorption dipoles, increases 2.9 +/- 0.6 A when the enzyme changes from the Na+ to the K+ conformation. Stopped-flow measurements of the change in fluorescence energy transfer efficiency with time when the doubly-labeled pump is mixed with Na+ or K+ demonstrate that the donor/acceptor pair reports the change between the E1 and E2 conformations of unphosphorylated enzyme. The observed first-order rate constant for the change in energy transfer efficiency depends sigmoidally on [K+] and inversely on [Na+], and both rate and amplitude data for the change in energy transfer efficiency can be fit with the same values of the rate and ion-dissociation constants as published data for the conformational change between E1 and E2 obtained by singly labeling the enzyme with fluorophores that report changes in protein microenvironment. The prerequisite for successfully measuring the distance change and equating the protein rearrangement with a step in the catalysis-transport cycle is that the donor by itself does not report the conformational change.
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