Characterization of Binding Affinity of Gas1 and Gas2 Mutant Proteins
Posted August 2nd, 2011 at 8:40 pm.
Abstract: Chloe Baumann
Mentor: Dr. Porello
The cell wall’s main component is a network of β-(1,3)-glucan chains, which are continuously remodeled to meet the needs of the cell in the different stages of cell development. The family of proteins known as Gas1-5, among other enzymes, are responsible for remodeling the cell wall. My work this summer will focus on comparing the substrate binding affinity of Gas1 and 2 as well as the substrate binding affinity of several structural modules. The effect of mutations in various regions of Gas1 will help determine the role that region plays in binding/catalytic activity. In the case of Gas1, several mutants of the protein will be studied as well, including Gas1 lacking a serine region (∆Ser), and two with mutations in the amino acid sequence of the enzyme that remove specific amino acids known to be located within the active site of the protein. The wild type (Gas1 and Gas2) and mutant (Gas1) enzymes will be purified using Ni2+- affinity chromatography and characterized using SDS-Page Gel Electrophoresis. Gas1/2 will then be studied for their substrate binding capabilities using fluorescence spectroscopy, a method of determining strength of binding abilities using the fluorescent properties found in specific amino acids. The results gained form the above studies will help determine how the mutations within Gas 1 impact the substrate-binding region of the protein structure.