Ph Effects on the Stability and Folding of Monomeric Caspases

Caspases are an ancient class of cysteinyl proteases that play an integral part in cell development and apoptosis as an evolutionarily conserved function. During apoptosis, the intracellular pH decreases from 7.4 to ~ 6.8 and cytosolic acidification affects the activation of caspase-3 and pH-dependent conformational changes affect the rate of auto maturation in the dimeric procaspase-3, which in turn influences the ability of this enzyme to execute the cell. Proteins are required to fold correctly and attain their native conformation which is vital for protein function and the survival of the organism. The protein folding landscape is sculpted through evolution and there exists an evolutionary selection for functional residues by selecting residues that stabilize the interactions to attain the native conformation while repressing the selection of non-native interactions that take place during folding. Through the course of evolution, how these proteins fold to the native conformation to contribute to the fitness of the organism while maintaining or altering the energy landscape that is guided by trade-offs between the stability and the activity of the protein remains elusive. To understand how the energy landscapes changed over time, we resurrected an ancestral caspase (existed about 650 milllion years ago) and we examined the folding pathways of caspases from Cnidarians, specifically corals. We studied the equilibrium unfolding of caspases from O.faveolata (species susceptible to disease) and P.astreoides (species resistant to disease) as a function of pH (between pH 3 and pH 10.5) to examine the changes in the context of stability and folding. The data shows that these monomeric proteins have a high secondary structure at the native state and unfold via a three-state process closer to the physiological pH (pH 6.5~pH8.5), via an intermediate state which is aggregation prone. However, outside of this pH range, the native protein loses its secondary structure and exists in an intermediate state which is stable. Due to this, these proteins undergo a two-state unfolding mechanism wherein, the protein transitions from an intermediate state to the unfolded state, reversibly. Overall, the free energy change for these primitive coral caspases is ~12.5kcal/mol, which is higher than what is observed for human caspases. This higher stability might be due to the decrease in the activity and the presence of certain stabilizing residues that are not observed in the human caspases.