| This dissertation deals with the evaluation of the validity of the current thermodynamic methods used to determine protein stability (with emphasis on the use of chemical denaturants), the biophysical characterization of protein denatured states, and the assessment of the effects of different cosolvents and cosolutes on protein conformational stability. The experimental techniques employed include the following biophysical techniques: calorimetry (differential scanning and isothermal titration calorimetry), potentiometry (pH titration of proteins to determine protonation states as a function of variables like denaturant concentration or pH), spectroscopy (fluorescence, CD, UV absorbance and 2nd derivative spectroscopy), analytical size exclusion chromatography, and dynamic light scattering; as well as standard molecular biology techniques (e.g., protein expression and purification, electrophoresis, etc.).; In this work, the different complexities involved in the accurate determination of unfolding free energy changes are investigated. Also, it is demonstrated that not all, if not most, protein denatured ensembles are structurally and thermodynamically equivalent (which is counter to what is usually assumed in the protein folding field), and that salts, denaturants, and other solutes/cosolvents can sometimes affect protein stability in wildly unexpected manners. |
