THE DEVELOPMENT OF BETA-ACTIN MUTANTS WITH ALTERED BINDING AFFINITES FOR ATP AND ADP

Abstract

A number of neurodegenerative diseases are accompanied by the formation of cofilin-actin rods in neural cells. These rods can cause synaptic dysfunction and block transport within neurites. The overall goal of this project is to create an optogenetic switch that incorporates both cofilin (an actin-binding protein) and actin for the study of cofilin-actin rod formation in neural cells undergoing oxidative stress. During the course of the work, a protocol has been created to express the protein [beta]-actin. Protein overexpression and purification, circular dichroism, mass spectrometry, and electrophoresis have been utilized to isolate and characterize [beta]-actin. Site-directed mutagenesis has been conducted to create a series of mutants to explore which residues are key for nucleotide binding. A number of challenges related to protein expression and stability of this protein precluded completion of these studies. As a result, we turned to mutants of optogenetic actin to study nucleotide binding in cells. The results from these studies indicate that point mutants in our actin construct, in conjunction with an optogenetic cofilin, can provide insight into the nucleotide binding state of actin under different oxidant concentrations.