| Engineering proteins with high stability (DeltaG), improved efficiency (kcat/Km), and better specificity (Kd) requires understanding of their thermodynamics of formation, stabilization, and function. Since thermodynamics of proteins has been conventionally studied in terms of response to thermal or chemical denaturants, there is increasing interest in understanding the effect of relatively less explored hydrostatic pressure (P). The pressure dependence of stability (DeltaG) is defined by the change in volume upon denaturation, Delta V = VU --VN = (∂DeltaG/∂P) T. Understanding the individual factors in total change in volume upon protein unfolding poses a fundamental problem, is highly biologically relevant in terms of life in the deep ocean as well as will allow us to develop rational approaches to engineer proteins with high pressure tolerance.;Temperature dependence of change in volume upon denaturation is defined by the changes in thermal expansivity (DeltaE), Delta E = (∂DeltaV/∂T) P = EU --EN. The knowledge of EN and EU, is as much important as knowledge VU and VN, as they provide insights related to hydration. PPC allows experimental measurement of the thermal expansion coefficient, alpha = E/V, of a protein in the native, alphaN ( T), and unfolded, alphaU (T), states as a function of temperature. Analysis of the thermal expansion coefficient profile, obtained over a broad temperature range, provides us the change in volume upon protein unfolding (DeltaV). Thus, not only can PPC thermally denature the protein but it also can obtain thermal expansion coefficients while doing so, two state analysis of which provides volumetric changes upon protein denaturation. Hence, PPC serves a dual purpose of measuring thermostability as well as pressure stability.;In this thesis document, I have extensively discussed a variety of molecular determinants of native state expansivity, established how the thermal expansivity obtained using PPC can be used as a probe to study protein compaction, characterized the volumetric properties of the secondary structural elements such as alpha-helix and beta- sheet using PPC, probed the molecular determinants of the change in volume upon protein unfolding (DeltaVvoid and DeltaVhydration) and finally, shed light on the protein volume paradox resolution and its biological implications for life at extreme environmental conditions. |
