UCSF

α-lytic protease (αLP), a bacterial serine protease of the chymotrypsin family, has evolved both kinetic stability and an extreme unfolding cooperativity in order to limit proteolysis. Trypsin, a well-studied metazoan homolog, is degraded at rates up to 100x faster than αLP even though it has approximately the same global unfolding rate. Previous experimental studies have implicated the interface between αLP's two domains as critical to the unfolding pathway and its cooperativity. To investigate this, I performed multiple high temperature molecular dynamics unfolding simulations on both αLP and trypsin. The simulations revealed a robust unfolding pathway that featured preferential disruption of the domain interface, primarily at three regions: the Domain Bridge, the C-terminal domain β-hairpin and cis-proline turn, and the N-terminus. I developed a metric for measuring global unfolding cooperativity, and it showed correctly that αLP unfolded cooperatively, while trypsin did not. I then applied an information-theory-derived measure of cooperativity developed by Voelz, based on pairs of contacts in the two proteins, to the simulations, allowing me to look at cooperativity at the residue level. By graphing the contact cooperativity as a network, I showed that the αLP network is significantly larger and more connected than that of trypsin, again showing a much higher global unfolding cooperativity. Using only the early parts of the simulations, the cooperativity network highlights contacts broken cooperatively around the transition state ensemble. These network graphs also identify residues that are key centers of cooperativity and if mutated, may disrupt αLP's unfolding cooperativity. Experimental studies are currently underway in the lab to test the hypotheses created from these simulations.