Exploring minimum free-energy pathway of GB1 dimerization in dilute and crowded solution.

The intracellular crowded environment plays a major role in driving the protein-protein association reaction that entails large conformational fluctuations. A detailed understanding of the crowding influence on protein association requires characterization of transient intermediates on a free energy landscape. In this work, we explore the free energy landscape of dimerization of protein GB1 in a dilute and crowded medium by employing advanced sampling techniques, such as metadynamics and parallel tempering. Dimerization proceeds via a single dominant pathway encountering few minima in dilute solutions. However, in presence of lysozyme crowders, the free energy landscape exhibits multiple minima and multiple barriers, providing alternative pathways for dimerization. The minimum free energy pathway indicates that dimerization starts by destabilizing the N-termini of monomers in both the cases. The population of the on-pathway intermediate states in dilute medium reveals the structural modulations in GB1 conformation that eventually lead to a final dimer-like state. The presence of lysozyme crowders stabilizes new intermediates, although no stable dimer is formed. The study highlights modification of dimerization pathway by attractive protein-crowder interactions.