Kinetic stability and temperature adaptation. Observations from a cold adapted subtilisin-like serine protease.

Abstract

Life on earth is found everywhere where water is found, meaning that life has adapted to extremely varied environments. Thus, protein structures must adapt to a myriad of environmental stressors while maintaining their functional forms. In the case of enzymes, temperature is one of the main evolutionary pressures, affecting both the stability of the structure and the rate of catalysis. One of the solutions Nature has come up with to maintain activity and stability in harsh environments over biological relevant timescales, are kinetically stable proteins. This thesis will outline work carried out on the kinetically stable VPR, a cold active subtilisin-like serine protease and discuss our current understanding of protein kinetic stability, temperature adaptation and our current hypothesis of the molecular interactions contributing to the stability of VPR. The research model that we have used to study these attributes consists of the cold active VPR and its thermostable structural homolog AQUI. The results discussed in this thesis will be on the importance of calcium, the role of prolines in loops, the role of a conserved N-terminal tryptophan residue and lastly primary observations on differences in active site dynamics between VPR and AQUI. A model is proposed of a native structure that unfolds in a highly cooperative manner. This cooperativity can be disrupted, however, by modifying calcium binding of the protein or via mutations that affect how the N-terminus interacts with the rest of the protein. The N-terminus likely acts as a kinetic lock that infers stability to the rest of the structure through many different interactions. Some of these interactions may be strengthened via proline residues, that seemingly act as anchor points that tend to maintain correct orientation between these parts of the protein as thermal energy is increased in the system. Our results give a deeper insight into the nature of the kinetic stability, the importance of cooperativity during unfolding of kinetically stable proteases, synergy between distant parts of the protein through proline mutations and how different calcium binding sites have vastly differing roles. The results provide a solid ground for continuing work in designing enzyme variants with desired stabilities and activities and improve our understanding of kinetically stable systems.