Protein complexation is a kinetic process, where the formation of an encounter (transient) complex precedes the formation of the final complex. We studied the nature of the encounter complex and transition state for binding both in buffer and in a crowded environment, and developed in silico tools to design faster and tighter binding protein complexes. We provided experimental evidence on how electrostatic forces increase the rate of association through the stabilization of the encounter complex without affecting the final docking rate. Combining experiments and computer simulations, we showed that only some of the encounters are fruitful while other are futile and that the transition state for binding can be specific or diffusive. We designed mutations that enhanced the fruitful and specific encounters, and by this increased their rate of association. Surprisingly, in a crowded environment the association and dissociation rate constants are almost as rapid as in water, apparently due to the occluded volume effect. Studying binding kinetics in real-time in living cells showed that binding is as fast in cells as in vitro and that electrostatic forces have similar effects also in cells. This validates many years of in vitro studies on this subject.
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