Date of Graduation

5-2015

Document Type

Thesis

Degree Name

Bachelor of Science

Degree Level

Undergraduate

Department

Chemistry & Biochemistry

Advisor

Adams, Paul D

Reader

Thallapuranam, Sureshkumar

Second Reader

McNabb, David

Third Reader

Brogi, Alessandro

Abstract

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.

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