The 8th Annual

NC State University

Undergraduate Summer Research Symposium

 

 

NC SEED Program - high school abstracts


Abstracts are listed in alphabetical order by the last name of the corresponding author.

 

 

 

 

 

Student Author(s): 

Melbourne, Victoria C.

Home Institution:

NCSU 

Program:

NC SEED Program – high school

Department(s):

Molecular and Structural Biochemistry

Research Mentor(s): 

Carla Mattos/Molecular and Structural Biochemistry

Gregory Buhrman/Molecular and Structural Biochemistry

Title of Presentation:

The Effect of a Residue 61 Mutation on the Structure of RAS GTPase

 

 

The Ras protein plays an important role in cellular growth and differentiation. In its active form, Ras-GTP binds to multiple downstream effector proteins activating a variety of signal transduction cascades. In its inactive, Ras-GDP form, binding to downstream effector proteins is impaired. One of the primary downstream effector proteins is Raf kinase. Point mutations in Ras at residues 12, 13, and 61 can result in cancer. Previous structural studies showed that the Q61L mutation induces a conformation in Ras that impairs hydrolysis of GTP to GDP. A hydrogen bond interaction between the backbone amide of leucine 61 and the catalytic water molecule appears to play a role in the reduced catalytic power. This project is aimed at investigating the role of this hydrogen bond by mutating glutamine 61 to proline, the only amino acid that lacks a backbone amide group, via site-directed mutagenesis. Site directed mutagenesis involved: primer design, PCR, and DNA transformation. The mutated protein was then over expressed in E. coli and purified by anion-exchange and gel filtration chromatography. The purified protein was then used in crystallization trials after exchanging the bound GDP nucleotide for a non-hydrolyzable GTP analog, GppNHp. The Q61P-Ras GppNHP was also tested for binding to Raf kinase to determine whether or not the mutated form of Ras will bind to its downstream effector Raf kinase.    

 

 

 

 

 

 

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