The proposed research program deals with fundamental questions regarding unfolding transitions in proteins and the interaction of specific ligands with proteins. The work will focus on two proteins, nuclease A and trp aporepressor, and certain site- directed mutants of these proteins. Dr. Eftink will study the cold (as well as high temperature) unfolding and pressure unfolding of relatively unstable mutants of nuclease. The goal is to evaluate and correlate various thermodynamic parameters for the unfolding of several mutants of nuclease in order to better understand the forces responsible for stabilizing proteins. The "structure" and dynamic characteristics of the various unfolding states (i.e., produced by low and high temperature, pressure, urea, low pH) will be studied by a combination of several techniques, including i) time-resolved fluorescence anisotropy to measure local motion (of the single trp residue), ii) resonance energy transfer between sites to obtain intramolecular distance information, iii) size exclusion chromatography to measure the overall hydrodynamic radius of the molecule, and iv) CD to observe any differences ins secondary structure. In addition to comparing the various unfolded states, the effect of the destabilizing mutations on the folded state of the proteins will be studied by some of the above methods and by v) room temperature phosphorescence to compare the rigidity of the environment of the trp residue, and vi) titration microcalorimetry to measure thermodynamic parameters for the binding of specific ligands to the protein.
