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ADP binding induces long-distance structural changes in the beta polypeptide of the chloroplast ATP synthase

Mills, D.A., Seibold, S.A., Squier, T.C., Richter, M.L.
Biochemistry 1995 v.34 no.18 pp. 6100-6108
H+/K+-exchanging ATPase, H-transporting ATP synthase, adenosine diphosphate, adenosine triphosphate, beef, binding sites, catalytic activity, chloroplasts, crystal structure, cysteine, dissociation, energy transfer, fluorescence, heart, information exchange, mitochondria, polypeptides
Binding of ADP to the beta polypeptide isolated from the catalytic F1 portion (CF1) of the chloroplast ATP synthase caused an increase of 10-20% in the steady state fluorescence intensity of fluorescent maleimides attached to the cysteine residue at position 63. Fluorescence lifetime distributions indicated that the beta polypeptide switched between two conformational states depending on the presence or absence of bound ADP. The fluorescence enhancement induced by ADP binding allowed a direct calculation of the dissociation constant for ADP of 0.7 micromolar. ATP did not cause a fluorescence enhancement but competed with ADP for binding to the same site. An apparent dissociation constant of 2 micromolar was obtained for ATP binding. Fluorescence resonance energy transfer experiments indicated that Cys63 is 42 angstroms away from the nucleotide binding site on the beta polypeptide, confirming a previous measurement [(Colvert, K. K., Mills, D. A., Richter, M. L. (1992) Biochemistry 31, 3930-3935]. Frequency domain fluorescence anisotropy measurements indicated that the beta polypeptide has an irregular, elongated shape which is in good agreement with the conformation found in the crystal structure of the beef heart mitochondrial F1 enzyme [Abrahams, J. P., Leslie, A. G. W., Lutter, R., & Walker, J. E. (1994) Nature 370, 621-628]. The rotational correlation time did not change significantly upon ADP binding, indicating that ADP did not induce a large change in the overall shape of the beta polypeptide. The results show that the nucleotide binding domain and the N-terminal domain of the beta polypeptide communicate with each other over a significant distance via conformational changes. This supports several other recent findings which have indicated that the N-terminal region of the beta polypeptide forms a site of contact with the a polypeptide and that this contact site is important for cooperative exchange of information between nucleotide binding sites during catalysis by CF1.