Abstract: Emily Rivers
Mentor: Silvia Porello
Gas1p is a glycoprotein found on the cell membrane of Saccharomyces cerevisiae, a species of yeast. The protein is anchored to the membrane through a glycophosphatidylinositol (GPI) moiety. The catalytic activity of Gas1 is essential for continuous cell wall synthesis and remodeling. The goal of my project is to determine the structural stability of the protein. Such proteins can be denatured, meaning that their structure can unfold under various conditions, such as the increase of temperature, presence of chemical denaturants, or varying the pH. This experiment manipulates these variables in order to determine the degree of denaturation of the protein as assessed by fluorescence spectroscopy.
The initial task is to purify a recombinant protein that lacks the GPI attachment site, which renders the protein soluble. We will also purify mutants, such as those that lack the Serine-rich region of the protein. The proteins will be purified utilizing affinity chromatography. After purification, we will measure the protein’s fluorescence emission through florescence spectroscopy. This will be run on the wild type and mutant Gas1p proteins that have been denatured by the manipulation of pH or temperature. If a change in fluorescence is observed upon denaturation, the two mutant proteins can be further analyzed to determine if the Serine-rich region is necessary for the stability of the protein. Continued research will determine the stability of Gas1p mutants as well as determining the effect of different protein regions in the structural stability of Gas1p.