Jason Westgeest - University of Alberta

Placement: University of Windsor
Supervisor: Dr. Art Szabo

Denaturation Studies on Tryptophan Mutants of Rat-Calmodulin

Calmodulin (CAM) is a protein which acts as a modulator of enzyme activity in a calcium dependent manner. By using Calmodulin mutants in which one amino acid has been selectively replaced by a single tryptophan, one can probe specific locations within the protein. The mutants I52W, I125W, I85W, L39W, and Y99W as well as the domains C-I125W, N-I52W, C-CAM, and N-CAM were thermally denatured from 10 C to 90 C and monitored using the techniques of Circular Dichroism, Fluorescence, and Time-Resolved Fluorescence. Both the apo (non-Calcium bound) and holo (Calcium bound) forms of the mutant proteins were studied. The integrated fluorescent intensity was monitored at different temperatures, and the melting temperature of the apo form occurred at temperatures that are 10 C to 15 C below those of the holo forms in the whole protein. Similar measurements were made on the N and C domains in an effort to learn more about the unfolding process and the manner in which the two domains communicate with one another. It has been found that CAM "is not the sum of its parts," (Sorensen and Shea (1998) Biochemistry 37, 4244 - 4253). The fluorescence is sensitive to local structural changes while the CD reports the global average secondary structure. Differences were noted between the two types of measurements performed.