The dissociation rate of the EcoRI-DNA-specific complex is linked to water activity.

In many respects, the dissociation rate constant of a DNA– protein or protein–protein complex is as important a physical parameter as the equilibrium constant. The regulation of most cellular activities and developmental control are dynamic rather than static processes. With many techniques, the successful physicochemical characterization of a complex depends critically on the lifetime of the complex during isolation or measurement. With present technologies, the very powerful, single molecule methods used for mapping the kinetic barriers of complex dissociation reactions require lifetimes on the order of minutes. We report here that the dissociation rate of the specific complex between the restriction nuclease EcoRI and its recognition DNA sequence is strongly dependent on water activity (in addition to its known dependence on salt activity). This observation means that the dissociation rate of complexes in the crowded conditions found within cells cannot be straightforwardly predicted from dilute solution measurements, even though salt, temperature, and pH conditions are fixed to those found in vivo. In addition, these results suggest a practical method to extend the lifetime of “weak” complexes sufficiently to perform biophysical and biochemical characterizations. The thermodynamic analysis of protein, peptide, and drug interactions with DNA has focused on the sensitivity of free energies to temperature, pH, and salt concentration (reviewed in Refs. 8–11). However, the displacement of water that should accompany specific complex formation as direct DNA–protein contacts replace DNA–water and protein–water interactions (reviewed in Refs. 12 and 13) means that binding energies will also depend on water activity. The number of water molecules released to the bulk solution in the process of DNA–protein complex formation can be measured from the sensitivity of the binding constant to bulk solution water activity. This procedure is analogous to measuring ion release through the dependence of binding constant on salt activity, or protonation through pH sensitivity. Water activity can be varied by adding neutral solutes that do not themselves directly affect the DNA–protein binding. This approach has been used to measure changes in hydration accompanying the DNA binding of several proteins: Escherichia coli gal repressor, E. coli CAP protein, Hin recombinase, Ultrabithorax and Deformed homeodomains, E. coli tyr repressor, EcoRI, and the Sso7d protein. Using an equilibrium competition approach, we showed previously that the free energy difference between complexes of the restriction nuclease EcoRI with nonspecific DNA and with the enzyme’s recognition sequence is linearly dependent on the change in water chemical potential of the solution with added osmolyte. This dependence translates into an additional ; 110 waters that are sequestered by the nonspecific complex relative to the specific complex at 20°C and ; 70 more waters at 4°C. This significant difference in retained waters between specific and nonspecific complexes is accompanied by a difference of ; 10 between the specific and nonspecific EcoRI DNA binding constants. The difference in hydration additionally was seen to be insensitive to the size and chemical nature of the solute used to change water activity for a wide variety of osmolytes. This result most probably implies that the water retained by the nonspecific complex is sequestered in a cavity at the DNA–protein interface that is sterically inaccessible to solutes.