University of Iceland, School of Engineering and Natural Sciences, Faculty of Physical Sciences
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.The Icelandic Research Fund [grant number 162977-051