Proton NMR studies of transforming and nontransforming H-ras p21 mutants
- 16 January 1990
- journal article
- research article
- Published by American Chemical Society (ACS) in Biochemistry
- Vol. 29 (2), 504-511
- https://doi.org/10.1021/bi00454a026
Abstract
One- and two-dimensional nuclear magnetic resonance spectroscopy (1D and 2D NMR) and site-directed mutagenesis were used to study the influence of mutations on the conformation of the H-ras oncogene product p21. No severe structural differences between the different mutants, whether they were transforming or nontransforming, could be detected. Initially, selective incorporation of 3,5-deuterated tyrosyl residues into p21 and 2D NMR were used to identify the resonances representing the spin systems of the imidazole rings of the three histidyl residues in the protein, of six of the nine tyrosyl rings, and of four of the five phenylalanyl rings. The spin systems of the phenyl rings of Phe28, Phe78, and Phe82 could be assigned by using mutant proteins, since no severe structure-induced spectral changes in the aromatic part of the spectra of the mutant proteins were detected. Sequence-specific assignments of the histidine imidazole resonances could be obtained by comparison of the distance information obtained by nuclear Overhauser enhancement spectroscopy (NOESY) experiments with the crystal structure. The change in the chemical shift values of the H1'' proton and the .alpha.-phosphate of the bound GDP in the NMR spectra of the p21(F28L) mutant and the 28-fold increase in the GDP dissociation rate constants of this mutant suggest a strong interaction between Phe28 and the p21-bound nucleotide. In solution, the p21-bound GDP .cntdot. Mg2+ has an anti conformation, and the phenyl ring of Phe28 is close to the ribose of the bound GDP .cntdot. Mg2+.This publication has 10 references indexed in Scilit:
- Yeast mutants temperature-sensitive for growth after random mutagenesis of the chromosomal RAS2 gene and deletion of the RAS1 gene.The EMBO Journal, 1988
- Mutations in the nucleotide binding loop of adenylate kinase of Escherichia coliBiochemistry, 1988
- ras GENESAnnual Review of Biochemistry, 1987
- Reactivity of a sulfhydryl group of the ras oncogene product p21 modulated by GTP binding.Journal of Biological Chemistry, 1986
- Isolation of ras GTP-binding mutants using an in situ colony-binding assay.Proceedings of the National Academy of Sciences, 1986
- Homologies Between Signal Transducing G Proteins and ras Gene ProductsScience, 1984
- Comparative biochemical properties of normal and activated human ras p21 proteinNature, 1984
- Homologies in the primary structure of GTP-binding proteins: the nucleotide-binding site of EF-Tu and p21.The EMBO Journal, 1984
- Studies on transformation of Escherichia coli with plasmidsJournal of Molecular Biology, 1983
- Exchange of Carbon-Bound Hydrogen Atoms ortho to the Hydroxyl Group in TyrosineScience, 1965