Date of Graduation
Bachelor of Science
Chemistry & Biochemistry
Adams, Paul D
Ras Homolog Enriched in Brain (RHEB) is a member of Ras GTPase family and plays an important role in regulation of cell growth and cell cycle proliferation. RHEB is regulated by cycling between active (GTP-bound) and inactive (GDP-bound) state. Guanidine Nucleotide Exchange Factors (GEFs) and GTPase-activationg proteins (GAPs) are the key regulators of RHEB, and it is of interest to closely study their interactions with RHEB. Towards that end, this project was designed to label RHEB with an environmentally sensitive extrinsic fluorophore, succinimidyl 6-(N-(7-nitrobenz-2-oxa-1.3-diazol-4-yl)amino)hexanoate (sNBD), to be used to monitor RHEB-protein interactions. Due to its extreme sensitivity to the surrounding environment, sNBD is expected to show changes in fluorescence when labeled RHEB reacts with other protein effectors. Histidine tagged RHEB was expressed using E. coli bacteria as host cells and was purified using Histidine affinity column chromatography. Time-dependent chymotrypsin digestion experiments were performed in order to compare digestion patterns of RHEB and sNBD-labeled RHEB. Using the purified protein, RHEB-sNBD binding procedure was optimized. A modified purification procedure had to be designed in order to meet the buffer environment and concentration requirement for the RHEB-sNBD binding procedure. The RHEB-sNBD binding experiment was determined to work best with 1:24 molar ratio of RHEB to sNBD. Successful RHEB-sNBD binding was performed using PBS buffer as the solvent condition for protein, and DMF as the solvent for sNBD.
Patel, Akash D., "Chymotrypsin Digestion Analysis to Characterize Site-Specific Incorporation of an Extrinsic Fluorescent Probe on a Ras-related Protein." (2015). Chemistry & Biochemistry Undergraduate Honors Theses. 5.