Use of the Crooks Fluctuation Theorem to Study the Inter-domain Folding Cooperativity of a Protein

The structures of proteins show a modular architecture comprised of discrete domains. Inter-domain communication regulates function and folding, while lack of coupling can lead to protein misfolding, aggregation and associated pathologies. However, how different regions communicate is still poorly understood. 
Using single-molecule optical tweezers, we induce the unfolding of regions of T4 lysozyme and monitor the effect on other regions not directly acted on. We determine the free energy landscape during mechanical unfolding using Crooks' fluctuation theorem, and show that the order of structural elements along the sequence determines what parts of the folding/unfolding landscape are explored. We speculate that proteins may have evolved to select certain topologies that increase coupling between regions to avoid areas of the landscape that lead to misfolding.