CONFORMATIONAL-CHANGES IN PROTEINS INDUCED BY DYNAMIC ASSOCIATIONS - A TRYPTOPHAN PHOSPHORESCENCE STUDY

Random collisions between macromolecules lead to dynamic associations (lengthy encounters) that in principle could affect their conformation and, in the case of enzymes, their binding and catalytic properties. Exploiting the unique sensitivity of the phosphorescence lifetime, tau , of Trp to the internal flexibility of globular proteins we probed th e perturbations induced in the structure of the coenzyme-binding domai n of alcohol dehydrogenase (LADH) and glyceraldehyde-3-phosphate dehyd rogenase (GraPDH) by the presence in solution of other dehydrogenases and of functionally unrelated proteins. With Trp314 of LADH, the resul ts emphasize that while tau is not affected by the concentration of LA DH itself, the addition of micromolar quantities of other proteins cau ses a distinct reduction in it. From the linear increase of 1/tau with protein concentration one obtains values for the apparent second-orde r Stern-Volmer rate constant that range between 2-200 x 10(3) M(-1), s (-1), decreasing 2-3-fold when ternary complexes of LADH with NADH or NAD(+) and inhibitors are involved. Similar effects were observed with Trp310 of GraPDH except that with sorbitol dehydrogenase as perturban t the increase of 1/tau is hyperbolic and governed by an apparent diss ociation constant of about 1 mu M. Finally, glycerol-3-phosphate dehyd rogenase, the strongest perturber of both LADH and GraPDH, has either no effect on lactic dehydrogenase from pig heart or induces a moderate lengthening of the triplet lifetime of the rabbit muscle enzyme. Beca use Stern-Volmer behaviour is typical also of diffusion-mediated quenc hing reactions, a parallel investigation with cysteine, cystine and N- acetyl-tryptophanamide demonstrated that among potential, protein-asso ciated, quenching moieties namely, -SH, -S-S- and indole groups, only the latter has rate constants approaching the magnitude of protein per turbants. Since considerable evidence rules out the predominance of su ch quenching reactions, these findings confirm a subtle form of commun ication between protein molecules in solution. The lack of specificity and the similar effects between dehydrogenases with right and wrong s tereospecificity for direct coenzyme transfer suggests that the pertur bations monitored are unrelated to this function.