Conformational changes in proteins induced by dynamic associations

Abstract

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, z, of Ti7 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 z 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 l/z with protein concentration one obtains values for the apparent second-order Stem-Volmer rate constant that range between 2-200 X lo3 M-' s-', 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 llt is hyperbolic and governed by an apparent dissociation constant of about 1 pM. 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 Stem-Volmer behaviour 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.Trp residues buried within globular proteins often display a long-lived phosphorescence emission even at ambient temperature in buffer [ l , 21. The lifetime, z, of the delayed emission shows a strong dependence on the mobility of the chromophores' environment and ranges between a few seconds within tight protein cores [l, 31 or rigid protein powders [4] and 15-20 ps [5] for the free chromophore in aqueous solutions. The dependence of z on the viscosity of the medium[6] thus provides a natural method for probing the local flexibility of protein cores. The dynamics of protein structure has proved to be particularly sensitive to even subtle changes in conformation of the macromolecule. In this respect Trp phosphorescence has been instrumental for highlighting conformational transitions in ...

Nanoparticle corona for proteins: mechanisms of interaction between dendrimers and proteins

“…Protein RTTP lifetimes depend strongly on oxygen removal [17][18][19][20][21][22]. The strong interaction of dendrimers with a protein surface can restrict the effective removal of oxygen from a protein globule [17][18][19][20][21][22], which results in a significant decrease of RTTP. As follows from data on zetapotentials, dendrimers have the strongest interaction with AP (Fig.…”

“…These marked differences in dendrimer-induced changes of secondary structure can be explained by the significant differences in stability/flexibility of the tertiary structure of the enzymes as monitored by tryptophan phosphorescence (Table, and refs. [20][21][22]). LDH tetramer and AST dimer are 1000 times more flexible than AP dimer (Fig.…”

“…AST is an enzyme that catalyzes the reversible transamination of the α-amino group from L-aspartate to α-ketoglutarate forming oxaloacetate and glutamate [14] (Table). The ligand-induced and catalytic conformational changes of the macromolecule are an integral feature of enzyme transamination [22][23][24]. Unliganded conformation is open and substrate-liganded is closed, in which a rigid body movement of the small domain closes the pocket of its active site.…”

“…AP from E. coli is a dimeric, non-glycosylated hydrolase enzyme responsible for removing phosphate groups from many types of molecules, including nucleotides, proteins, and alkaloids [12]. It has an extremely stable structure [21][22]25], and its active site is buried at 11 Å inside it [17,25]. Human and yeast APs are 10 to 20 fold more active than the E. coli AP, but they are much less heat stable, and show a shift in the pH versus activity profile at higher pH [25].…”

“…All phosphorescence decays after subtraction of the background were analyzed in terms of a sum of exponential components by a non-linear least squares fitting algorithm, using the software provided by Edinburgh Analytical Instrumentation (Edinburgh Instruments, UK). τ average was calculated as τ = α 1 τ 1 +α 2 τ 2 +α 3 τ 3 , α i and τ i -corresponding contribution and lifetime of RTTP decay [18][19][20][21][22]. Statistical analysis.…”