Analysis of equilibrium and kinetic measurements to determine thermodynamic origins of stability and specificity and mechanism of formation of site-specific complexes between proteins and helical DNA.

The concentration and nature of the electrolyte are key factors determining (1) the equilibrium extent of binding of oligocations or proteins to DNA, (2) the distribution of bound protein between specific and nonspecific sites, and (3) the kinetics of association and dissociation of both specific and nonspecific complexes. Salt concentration may therefore be used to great advantage to probe the thermodynamic basis of stability and specificity of protein-DNA complexes, and the mechanisms of association and dissociation. Cation concentration serves as a thermodynamic probe of the contributions to stability and specificity from neutralization of DNA phosphate charges and/or reduction in phosphate charge density. Cation concentration also serves as a mechanistic probe of the kinetically significant steps in association and dissociation that involve cation uptake. In general, effects of electrolyte concentration on equilibrium constants (quantified by SKobs) and rate constants (quantified by Skobs) are primarily cation effects that result from the cation-exchange character of the interactions of proteins and oligocations with polyanionic DNA. The competitive effects of Mg2+ or polyamines on the equilibria and kinetics of protein-DNA interactions are interpretable in the context of the cation-exchange model. The nature of the anion often has a major effect on the magnitude of the equilibrium constant (Kobs) and rate constant (kobs) of protein-DNA interactions, but a minor effect on SKobs and Skobs, which are dominated by the cation stoichiometry. The order of effects of different anions generally follows the Hofmeister series and presumably reflects the relative extent of preferential accumulation or exclusion of these anions from the relevant surface regions of DNA-binding proteins. The question of which anion is most inert (i.e., neither accumulated nor excluded from the relevant regions of these proteins) remains unanswered. The characteristic effects of temperature on equilibrium constants and rate constants for protein-DNA interactions also serve as diagnostic probes of the thermodynamic origins of stability and specificity and of the mechanism of the interaction, since large changes in thermodynamic and activation heat capacities accompany processes with large changes in the amount of water-accessible nonpolar surface area.(ABSTRACT TRUNCATED AT 400 WORDS)