This last semester, I hope to isolate the hsfi1 D10 construct for future dynamic studies, where we hope to gain a deeper understanding of the dynamics involved during complex formation of the hsfi1 construct with hcentrins. The isolation part of the project is nearly done. Nonetheless, there is great difficulty in maintaining the isolated construct soluble. Introduction of detergents to force solubility can interfere with our subsequent 2D-correlational spectroscopy studies. We hope hsfi1-hcentin complex formation will aid in solubility to the extent necessary for the dynamic studies we’re currently interested in conducting. We will also be performing 2D-COS studies for several hcentrin mutants using external temperature perturbations. We will use these studies to compare the order of events taking place during protein unfolding and see how each one varies with respect to the other. Analogous experiments will be conducted for the hsfi1-hcentrin complexes as soon as the hsfi1 constructs of interest are isolated.
As I had mentioned in the last time I posted, once the hsfi1 D10 construct is isolated and an official protocol is established, we will then endeavor purifying the hsfi1 D10-12 construct. The latter, as oppose to the former, is of utter importance, for it will allow us to follow the binding mechanism of, not only a single hcentrin with hsfi1, but possibly two hcentrins. Furthermore, we believe there might also we present an interaction between neighboring hcentrins once they bind to hsfi1. We hope 2D-correlational spectroscopy studies will allow us to confirm whether these interactions are present, and in which order of events they’re taking place.
