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 the perturbations induced in the structure of the coenzyme-binding domain of alcohol dehydrogenase (LADH) and glyceraldehyde-3-phosphate dehydrogenase (GraPDH) by the presence in solution of other dehydrogenases and of functionally unrelated proteins. With Trp314 of LADH, the results emphasize that while tau is not affected by the concentration of LADH itself, the addition of micromolar quantities of other proteins causes a distinct reduction in it. From the linear increase of 1/tau with protein concentration one obtains values for the apparent second-order 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 perturbant the increase of 1/tau is hyperbolic and governed by an apparent dissociation constant of about 1 microM. Finally, glycerol-3-phosphate dehydrogenase, 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. Because Stern-Volmer behavior is typical also of diffusion-mediated quenching reactions, a parallel investigation with cysteine, cystine and N-acetyl-tryptophanamide demonstrated that among potential, protein-associated, quenching moieties namely, -SH, -S-S- and indole groups, only the latter has rate constants approaching the magnitude of protein perturbants. Since considerable evidence rules out the predominance of such quenching reactions, these findings confirm a subtle form of communication between protein molecules in solution. The lack of specificity and the similar effects between dehydrogenases with right and wrong stereospecificity for direct coenzyme transfer suggests that the perturbations monitored are unrelated to this function.
Gabellieri
E
E
C.N.R., Istituto di Biofisica, Pisa, Italy.
Strambini
G B
GB
D000426:Alcohol Dehydrogenase / Q000737:chemistry; D000818:Animals; D004926:Escherichia coli / Q000201:enzymology; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000737:chemistry; D005993:Glycerolphosphate Dehydrogenase / Q000737:chemistry; D007064:L-Iditol 2-Dehydrogenase / Q000737:chemistry; D007770:L-Lactate Dehydrogenase / Q000737:chemistry; D008163:Luminescent Measurements; D011487:Protein Conformation; D011817:Rabbits; D012996:Solutions; D013552:Swine; D013696:Temperature; D014364:Tryptophan / Q000737:chemistry*
D012996:Solutions; D014364:Tryptophan; D005993:Glycerolphosphate Dehydrogenase; D000426:Alcohol Dehydrogenase; D007064:L-Iditol 2-Dehydrogenase; D007770:L-Lactate Dehydrogenase; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases