Functions of the Subunits of the Chloroplast ATP Synthase

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The structure, mechanism and regulation of the ATP synthases of chloroplast, mitochondrial, and bacterial membranes are major problems in biochemistry. This proposal deals with chloroplast purification of the subunits of CFo, the integral membrane portion of the chloroplast ATP synthase, in active form, and the reconstituted of proton translocation and the binding of CF 1, the extrinsic, catalytic part of the synthase. Fluorescence resonance energy transfer will be used to extend the extensive structural map obtained for CF1 to CFo. The binding of nucleotides to CF1 in relation to initial rates of catalysis will be examined. Calorimetric analyses are proposed that should give information about ATP-induced conformational changes in CF1 as well as about the role of nucleotide binding in stabilizing the enzyme. Structural mapping of a fourth nucleotide binding site will be carried out. The role of the epsilon subunit of CF1 will be examined. The binding of epsilon to CF1 deficient in this subunit will be studied by fluorescence and by isothermal titration calorimetry.