1. Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-0520, USA

Correspondence to: G. Marius Clore¹ Correspondence and requests for materials should be addressed to G.M.C. (Email: mariusc@intra.niddk.nih.gov).

Kinetic data on a number of protein–protein associations have provided evidence for the initial formation of a pre-equilibrium encounter complex that subsequently relaxes to the final stereospecific complex¹. Site-directed mutagenesis^{2, 3, 4} and brownian dynamics simulations^{5, 6, 7} have suggested that the rate of association can be modulated by perturbations in charge distribution outside the direct interaction surfaces. Furthermore, rate enhancement through non-specific binding may occur by either a reduction in dimensionality⁸ or the presence of a short-range, non-specific attractive potential⁹. Here, using paramagnetic relaxation enhancement, we directly demonstrate the existence and visualize the distribution of an ensemble of transient, non-specific encounter complexes under equilibrium conditions for a relatively weak protein–protein complex between the amino-terminal domain of enzyme I and the phosphocarrier protein HPr. Neither the stereospecific complex¹⁰ alone nor any single alternative conformation can account fully for the intermolecular paramagnetic relaxation enhancement data. Restrained rigid-body simulated annealing refinement against the paramagnetic relaxation enhancement data enables us to obtain an atomic probability distribution map of the non-specific encounter complex ensemble that qualitatively correlates with the electrostatic surface potentials on the interacting proteins. Qualitatively similar results are presented for two other protein–protein complexes.

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