CONFORMATIONAL DYNAMIC PROPERTIES OF WATER-SOLUBLE COUPLING FACTOR OF PHOTOPHOSPHORYLATION STUDIED BY SPIN-LABELING

The conformational dynamics of the isolated latent and activated water-soluble coupling factor of photo-phosphorylation (CF1) was studied applying preferential spin-labelling of -SH, -COOH and -NH2 amino acid side-chain groups and EPR spectra analysis permitting a quantitative discrimination between slow macromolecular rotation (τc) and fast anisotropic spin-label motion (S), as well as estimation of the relative distance between the nitroxide radicals attached to the protein. The labelling stoichiometry in most cases was found to depend on the enzyme activation state and medium pH. The protein correlation time (τc) of the non-activated CF1 (tempoylmaleimide and tempoylisothiocyanate derivatives) was estimated to be 150-159 ns, and that of the activated CF1 193-197 ns (data interpreted as CF1 hydrodynamic volume enlargement after activation). The significant τc differences registered after ligation of the activated enzyme with ATP and Ca2+-ATP are assigned to both segmental flexibility and hydration changes. The correlation time (τc = 10 ns) obtained for tempoylcarbodiimide CF1 derivative (probable labelling of β-subunit active site), together with the low spin-label restriction (S = 0.78) indicates a high degree of independent motion of the β-subunits with respect to that of the macromolecule, and/or microstructure flexibility in the vicinity of -COOH groups. When two carboxyl groups were modified (in the absence of Me2+ in the medium) the distance between the nitroxide radicals was calculated to be 45 Å, thus indicating that -COOH groups on different β-subunits were labelled. Conformational rearrangements (involving protein shadowing of several -NH2 groups) were registered when the EPR spectra of latent and activated CF1 tempoylisothiocyanate derivatives were analyzed by different techniques. © 1990.