Analysis of the small GTP binding protein Rac2
Snodgrass, Meagan Alyssa.
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Human neutrophils serve as the first line of immune defense against invading pathogens. In many cells, including neutrophils, Rac proteins are key regulators of many diverse cellular functions through their affects on cytoskeletal organization and in neutrophils, the NADPH oxidase complex, a critical mechanism for host defense. The primary purpose of this research is to examine the possibility that Rac2 interacts directly with the actin cytoskeleton. A different small GTP binding protein, Rap2 has been shown to interact with actin through direct binding. To test our hypothesis, GST-Rac2 and GST-Rac2 R120E protein was expressed in E. coli. Rac2 was then purified and used for phage display mapping to identify potential binding epitopes on actin. Phage display analysis, which has been used to map binding epitopes on other proteins, was conducted on GST-Rac2 and GST-Rac2 R120E affinity columns. Peptide sequences from phage display on both proteins were mapped to the amino acid sequence of human actin using the program FINDMAP. This program can identify discontinuous epitope regions by permitting large gaps in the target sequence during alignment. FINDMAP results were visualized using Swiss PDB Viewer, a publicly available protein modeling program and workbench. Several possible binding epitopes for Rac2 were identified on the surface of actin. Purified GST-Rac2 was shown to bind GTPã [35S] selectively, but GST-Rac2 R120E did not bind GTPã [35S]. Fluorescence spectroscopy results suggested that the structural conformation of the purified wild type and mutant protein were similar. Protein characterization shows that while only GST-Rac2 can bind nucleotide, both proteins are structurally similar. In addition to the purification of recombinant protein expressed in E. coli, we generated vectors to express c-myc tagged Rac2 and Rac2 R120E in mammalian cell lines. Preliminary fluorescence microscopy data shows that indeed c-myc tagged Rac2 and Rac2 R120E are expressed using an inducible transfection system. Future studies will determine the effect of Rac2 R120 E on the actin cytoskeleton in mammalian cells. Taken with previous data suggesting that Rac2 and Rac2 R120E binds actin, this research strengthens the argument that Rac2 and actin interact in a direct manner.