Developing Biochemical Probes for a Required Activation Step of Arp2/3 Complex

30 pages. A thesis presented to the Department of Human Physiology and Biology and the Clark Honors College of the University of Oregon in partial fulfillment of the requirements for degree of Bachelor of Science, Spring 2016.The Arp2/3 complex is a branched actin filament nucleator consisting of seven protein subunits. The Arp2/3 complex plays a crucial role in dynamic actin assembly, which is required for important cellular processes such as cell motility, cell division, chemotaxis, and phagocytosis. Previous studies have shown that Arp2/3 complex requires actin monomers, actin filaments, and nucleation-promoting factors such as W ASp for activation. However, it is still unclear how these factors cooperate to activate the Arp2/3 complex due to the lack of high resolution crystal structure of the active . ... 'iit. Arp2/3 complex. This study seeks to develop biochemical probes to investigate th;-· structural changes that activate the Arp2/3 complex. We hypothesize that there will be an additional conformational change after Arp2 and Arp3 subunits form a pseudo dimer that allows the Arp2/3 complex to bind to the actin filaments and nucleate actin polymerization. We have developed a crosslinking assay with Saccharomyces cerevisiae Arp2/3 complex that contains double cysteine mutations at the nucleotide cleft to test for the new proposed conformational change in the Arp2/3 complex. I found that R64C/Q214C mutant successfully crosslinked with bis-maleimidoethane (BMOE), and the presence of ATP significantly decreased the crosslinking reaction rate. However, this study only shows the preliminary data and further studies are needed to investigate the role of actin filaments in the activation of Arp2/3 complex