title: Electrostatic effects of mutations of Ras glutamine 61 measured using vibrational spectroscopy of a thiocyanate probe.
abstract: Mutations of human oncoprotein p21(Ras) (hereafter Ras) at glutamine 61 are known to slow the rate of guanosine triphosphate (GTP) hydrolysis and transform healthy cells into malignant cells. It has been hypothesized that this glutamine plays a role in the intrinsic mechanism of GTP hydrolysis by interacting with an active site water molecule that electrostatically stabilizes the formation of the charged transition state at the γ-phosphate during hydrolysis. We have tested the interactions between amino acids at this position and water by measuring changes in the electrostatic field experienced by a nitrile probe positioned near Ras Q61 using vibrational Stark effect (VSE) spectroscopy. We mutated this glutamine to every amino acid except cysteine and proline and then incubated these mutants with a Ral guanine nucleotide dissociation stimulator (Ral) containing the I18C mutation that was chemically labeled with a thiocyanate vibrational spectroscopic probe. The formation of the docked Ras Q61X-labeled Ral complex was confirmed by measurement of the dissociation constant of the interaction. We measured the absorption energy of this nitrile to determine any differences in electrostatic environment in the immediate vicinity of the thiocyanate probe between wild type and mutants of Ras. For each Ras Q61X mutant, we correlate the change in electrostatic field at position 61 with the solvent accessible surface area of polar components of the mutant side chain determined from a Boltzmann-weighted ensemble of structures, as well as the residue's hydration potential. These results support the hypothesis that the role of Ras Q61 is to stabilize water in or near the active site during GTP hydrolysis. The substantial effect that nonpolar side chains of Ras Q61X have on the absorption energy of the thiocyanate must be investigated with further experiments.
authors.affiliation: Department of Chemistry and Biochemistry, Institute for Cell and Molecular Biology, and Center for Nano- and Molecular Science and Technology, The University of Texas at Austin, 1 University Station, A5300, Austin, Texas 78712, United States.
authors.forename: Amy J
authors.initials: AJ
authors.lastname: Stafford
authors.forename: David M
authors.initials: DM
authors.lastname: Walker
authors.forename: Lauren J
authors.initials: LJ
authors.lastname: Webb
mesh_terms: D005973:Glutamine / Q000737:chemistry*; D056004:Molecular Dynamics Simulation; D009154:Mutation; D013057:Spectrum Analysis / Q000379:methods*; D055672:Static Electricity; D013861:Thiocyanates / Q000737:chemistry*; D018631:ras Proteins / Q000737:chemistry* / Q000235:genetics
chemical_list: D013861:Thiocyanates; D005973:Glutamine; D018631:ras Proteins