CusF, a protein model for folding and stability characterization
Authors:Jose Castellon, Ronandro De Guzman, Maria Gil
Mentor:Blake Gillespie , Associate Professor of Chemistry, California State University Channel Islands
Understanding protein stability would help shed light on misfolding events that cause many diseases, and understanding the up-stabilization caused by protein-ligand interaction opens possibilities for treatment of diseases caused by protein unfolding. In our experiments we use CusF, a small periplasmic Cu (I) and Ag (I) chaperone protein from E. coli, as our model to study the mechanism of protein folding. We characterized CusF’s conformational equilibrium and dynamics in the absence and presence of its ligand Ag (I) by employing two methods: hydrogen exchange NMR spectroscopy and fluorescence spectroscopy. Fluorescence-monitored chemical denaturations were conducted for the apo- and holo-forms over a broad temperature range, which allowed construction of stability curves for both forms, and indicated ligand binding results in a sharp decrease in CusF’s heat capacity of unfolding. Hydrogen-deuterium exchange NMR was used to measure local protein stability by using exchange rates of hydrogen bonded to the nitrogen atoms of the peptide chain backbone of our protein model. We find the ligand-dependent stabilization of the protein is highly localized, but radiates outward from the ligand binding site to distant regions of the molecule. We are investigating whether the ligand dependent stabilization of the protein derives from retention of residual structure in the denatured state.