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Electric field measurements in the active site of Ras
     
Ras active site
 
 

Previous work in our lab has established vibrational stark effect spectroscopy (VSE spectroscopy) as a technique for measuring changes in the local electric field in complex organized systems such as proteins. The goal of my project is to use VSE spectroscopy to study the electric field in the active site of the small GTPase Ras. Ras is an important component of growth factor signaling pathways in cells and is estimated to be mutated in ~30% of human cancers. When bound to the nucleotide guanosine triphosphate (GTP), Ras adopts a conformation that allows it to interact with and activate downstream signaling proteins, notably the protein kinase Raf. Hydrolysis of GTP to GDP results in a conformational change in Ras that prevents interactions with Raf and effectively turns off the signaling pathway. Nucleotide hydrolysis is enhanced by the binding of accessory proteins called GTPase activating proteins (GAPs), and oncogenic mutations in Ras exert their effect by interfering with GAP function, thereby trapping Ras in the active GTP-bound state. The binding of GAP proteins to Ras enhances hydrolysis by ~5 orders of magnitude. The crystal structure of the Ras:GAP complex shows that the GAP protein inserts an arginine residue into the active site of Ras in close proximity to the phosphates of GTP (see figure), and mutagenesis has shown that this so-called arginine finger is critical for enhancement of GTPase activity. Computational work has suggested that a large part of the GAP-mediated rate enhancement is due to electrostatic stabilization of the transition state of the hydrolysis reaction by the arginine finger. In order to test this hypothesis experimentally I am using vibrational stark effect probes in Ras and RasGAP proteins to measure the contribution of the arginine finger to the local electric field in the active site of Ras. I am also planning to use time-resolved FTIR measurements to follow changes in the electric field in the active site of Ras as the protein undergoes nucleotide hydrolysis.

 
         
  Recent Publications      
 

"Electric Fields at the Active Site of an Enzyme: Direct Comparison of Experiment with Theory ", Ian T. Suydam, Christopher D. Snow, Vijay S. Pande, and Steven G. Boxer, Science, 313, 200-204 (2006). [pdf]

"Vibrational Stark Effects Calibrate the Sensitivity of Vibrational Probes for Electric Fields in Proteins", Ian T. Suydam and Steven G. Boxer, Biochemistry, 42, 12050-12055, (2003). [pdf]

 
 
     
The Boxer LaboratoryStanford UniversityDepartment of Chemistry • 380 Roth Way, Stanford, California, 94305-5012 • (650) 723-4482
Questions about this website may be directed to Debra Frank. • Website updated March 2012.