Stephanie Truhlar

Like most extracellular bacterial proteases, Streptomyces griseus protease B (SGPB) and a-lytic protease (aLP) are synthesized with covalently attached pro regions necessary for their folding. The aLP folding mechanism has been previously characterized, revealing a very striking folding landscape with many functional implications (see also I. Pro region assisted folding and kinetic stability). Characterization of the folding free energy landscape of SGPB and comparison with the folding landscapes of aLP and trypsin, a mammalian homologue that folds independently of its zymogen peptide, has yielded many insights into the development of their distinct folding mechanisms. In contrast to the thermodynamically stable native state of trypsin, SGPB and aLP fold to native states that are thermodynamically marginally stable or unstable, respectively. Instead, their apparent stability arises kinetically, from unfolding free energy barriers that are both large and highly cooperative. The unique unfolding transitions of SGPB and aLP extend their functional lifetimes under highly degradatory conditions beyond that seen for trypsin; however, the penalty for evolving kinetic stability is remarkably large in that each factor of 2.4–8 in protease resistance is accompanied by a cost of ~105 in the spontaneous folding rate and ~5–9 kcal/mol in thermodynamic stability. These penalties have been overcome by the co-evolution of increasingly effective pro regions to facilitate folding. Despite these costs, kinetic stability appears to be a potent mechanism for developing native-state properties that maximize protease longevity.

I am currently pursing two approaches with the goal of gaining a mechanistic understanding of kinetic stability. The first approach is performing a detailed thermodynamic analysis of the SGPB unfolding mechanism. Comparison of the thermodynamics of the unique unfolding transitions of aLP and SGPB, as well as a thermophilic homolog being characterized by Brian Kelch, should elucidate the determinants of these distinctive, functionally important unfolding free energy barriers. Preliminary data shows that the enthalpy and entropy of the unfolding transitions are similar for aLP and SGPB. However, there appears to be a significant difference in the change in heat capacity upon unfolding. Furthermore, I am trying to determine the structural origins of the unique energetics of these proteases through mutagenesis. I have designed a number of aLP -SGPB chimeras and an analysis of their folding properties is underway.