ADP BINDING INDUCES LONG-DISTANCE STRUCTURAL-CHANGES IN THE BETA-POLYPEPTIDE OF THE CHLOROPLAST ATP SYNTHASE

Binding of ADP to the beta polypeptide isolated from the catalytic F-1 portion (CF1) of the chloroplast ATP synthase caused an increase of 1 0-20% in the steady state fluorescence intensity of fluorescent maleim ides attached to the cysteine residue at position 63. Fluorescence lif etime distributions indicated that the beta polypeptide switched betwe en two conformational states depending on the presence or absence of b ound ADP. The fluorescence enhancement induced by ADP binding allowed a direct calculation of the dissociation constant for ADP of 0.7 mu M. ATP did not cause a fluorescence enhancement but competed with ADP fo r binding to the same site. An apparent dissociation constant of 2 mu M was obtained for ATP binding. Fluorescence resonance energy transfer experiments indicated that Cys63 is 42 Angstrom away from the nucleot ide binding site on the beta polypeptide, confirming a previous measur ement [(Colvert, K. K., Mills, D. A., Richter, M. L. (1992) Biochemist ry 31, 3930-3935]. Frequency domain fluorescence anisotropy measuremen ts indicated that the beta polypeptide has an irregular, elongated sha pe which is in good agreement with the conformation found in the cryst al structure of the beef heart mitochondrial F-1 enzyme [Abrahams, J. P., Leslie, A. G. W., Lutter, R., & Walker, J. E. (1994) Nature 370, 6 21-628]. 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 polype ptide communicate with each other over a significant distance via conf ormational changes. This supports several other recent findings which have indicated that the N-terminal region of the beta polypeptide form s a site of contact with the a polypeptide and that this contact site is important for cooperative exchange of information between nucleotid e binding sites during catalysis by CF1.