Summary:
In Protein Science, one of the major research challenges has been the
correlation of the thermodynamic stability with structural elements, leading to
specific molecular functionalities in vivo. Advancing our understanding of this
association has helped the development of a series of applications in the
fields of biotechnology, nanotechnology, the envi-ronment and health. In this
Thesis, heat-induced as well as chemical denaturation is em-ployed in order to
accurately measure the thermodynamic stability of (a) lysozyme in solutions
containing various concentrations of NaCl and Na2SO4, two salts that promote
the crystallization of the molecules and of Li2SO4 and (NH4)2HPO4, two salts
that obstruct crystallization and (b) the catalytic domain of the psychrophilic
chitinase-60, a (b/a)8-barrel fold, which is of great importance in protein
engineering mainly for its function in polymer hydrolysis. Both the thermal and
the chemical denaturation explore the transition between the native, functional
and fully folded state of the protein and the non-functional, denatured,
partially or fully unfolded state. High-accuracy adiabatic differential
scanning calorimetry, circular dichroism (CD) and fluorescence spectroscopy
have all been used to systematically study the structural transitions. In the
case of lysozyme in solution with crystallization-promoting salts, the
thermodynamic stability is found to increase with the salt concentration, which
can be directly attributed to the decreasing entropy of the native state. In
this case the higher degree of organization of the native state in solution
leads to a higher entropic-gain upon crystallization which is the driving force
of the process. In the second part of the Thesis, the catalytic domain of
chitinase-60 is shown to exhibit a thermodynamic autonomy as well as an
extraordinary resistivity to chemical denaturants. The thermodynamic stability
was measured ΔG ~ 7 kcal/mol, while both thermal and chemical unfolding
processes were characterized by high kinetic barriers. The (b/a)8-barrel fold
of chitinase-60 is thus found to exhibit a “plug-and-play-domain” profile which
combined to its chemical resistivity establishes it as a very attractive
molecule for biotechnological environmental applications .
Keywords:
Proteins, Thermodynamic Stability, Crystallization, Differential Scanning Calorimetry, Fluorescence Spectroscopy
