Kinetics and mechanism of dissociation of cooperatively bound T4 gene 32 protein-single-stranded nucleic acid complexes. 2. Changes in mechanism as a function of sodium chloride concentration and other solution variables.

The dissociation kinetics of bacteriophage T4 coded gene 32 protein-single-stranded nucleic acid complexes have been examined as a function of monovalent salt concentration, temperature, and pH in order to investigate the details of the dissociation of cooperatively bound protein. Fluorescence stopped-flow techniques were used, and irreversible dissociation was induced by a combination of [NaCl] jumps and mixing with excess nucleic acid competitor. This made it possible to directly investigate the irreversible dissociation process over a wide range of NaCl concentrations [e.g., from 50 mM to 0.60 M for the gene 32 protein-poly(A) complex], in the absence of reassociation. Over the entire salt range, the only dissociable species observed is the singly contiguously bound gene 32 protein which dissociates from the ends of protein clusters. However, the [NaCl] dependence of the dissociation rate constant suggests that two competing pathways exist for dissociation of cooperatively bound gene 32 protein from the ends of protein clusters. At high monovalent salt concentrations, dissociation is dominated by a single-step process, with log ke/log [NaCl] = 6.5 +/- 0.5; i.e., the dissociation rate constant increases with increasing NaCl concentration due to the uptake of approximately six monovalent ions upon dissociation. This indicates that singly contiguous protein dissociates directly into solution. However, at much lower [NaCl] the data suggest that gene 32 protein, when bound at the end of a protein cluster, dissociates by first sliding off the end to form a noncooperatively bound intermediate which subsequently dissociates. A quantitative model which incorporates the sliding pathway [Berg, O. G., Winter, R. B., & von Hippel, P. H. (1981) Biochemistry 20, 6929-6948] in the dissociation mechanism fits the data reasonably well and suggests that noncooperatively bound monomers of gene 32 protein may be capable of one-dimensional translocation along single-stranded nucleic acids as suggested by independent kinetic data on the association reaction [Lohman, T. M., & Kowalczykowski, S. C. (1981) J. Mol. Biol. 152, 67-109]. It is also observed that both the absolute dissociation rate constant for T4 gene 32 protein and its salt dependence are sensitive to the average molecular weight and polydispersity of the nucleic acid sample used. This is a general phenomenon exhibited by proteins that bind to nucleic acids in a highly cooperative manner.