Role of Electrostatic Interactions in Transient Encounter Complexes in Protein−Protein Association Investigated by Paramagnetic Relaxation Enhancement

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At equilibrium, the complex between the N-terminal domain of enzyme I (EIN) and HPr consists of a stereospecific complex in rapid exchange with a minor population (≤10%) of an ensemble of nonspecific encounter complexes. We have investigated the effect of ionic strength on the magnitude of the intermolecular paramagnetic relaxation enhancement (PRE) rates using HPr labeled with EDTA−Mn2+ at two different sites, E5C and E25C. The magnitude of PREs attributable to the stereospecific complex shows only a small dependence on salt concentration and can be accounted for by the dependence of the overall equilibrium dissociation constant on salt concentration, as determined by isothermal titration calorimetry, resulting, under the conditions of the NMR experiments, in a small decrease in the population of bound EIN as the salt concentration is increased from 0 to 0.5 M NaCl. The magnitude of intermolecular PREs originating from the ensemble of nonspecific encounter complexes, however, is much more sensitive to salt concentration, implying that the population of nonspecific encounter complexes is significantly more affected by ionic strength than that of the stereospecific complex. These results demonstrate the importance of electrostatic interactions in the formation of the ensemble of nonspecific encounter complexes and are fully consistent with Debye−Hückel theory since the less compact interfacial packing for the nonspecific encounter complexes relative to the stereospecific complex permits more effective screening of intermolecular electrostatic interactions by ions in solution.