BIOMAT Database Logo BIOMAT Database Logo

Semisynthesis of H-Ras with a glutamic acid methylester at position 61. 2008

Jelena Milić, and Ralf Seidel, and Christian F W Becker, and Roger S Goody, and Martin Engelhard
Max-Planck-Institut für Molekulare Physiologie, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany.

The mechanism of the guanosine triphosphate (GTP) hydrolysis reaction of small G-proteins such as Ras is generally understood; however, some important molecular details are still missing. One example concerns the role of Gln61 in the catalysis of the GTP hydrolysis reaction. This amino acid is frequently mutated in oncogenic Ras leading to constitutively active variants of the protein. To elucidate the role of Gln61, subtle structural changes were introduced at this position by exchanging the natural occurring glutamine against a glutamic acid methyl ester (GluOme). Thereby the H-bond donor properties of this residue are changed and analysis of the GTP hydrolysis reaction can provide information on the function of the native carboxamide moiety. Using a semisynthetic approach, Ras(1-166)Gln61GluOMe was synthesized by sequential native chemical ligation of three unprotected peptide segments. Peptides Ras(1-50) and Ras(51-79)Gln61GluOMe were synthesized using Boc chemistry. The C-terminal peptide Ras(80-166) was expressed in E. coli. Initial tests of this semisynthetic strategy were performed by synthesis of the N- and C-terminally truncated protein variant Ras(39-101)Gln61GluOMe. The identified optimal reaction conditions were then applied to the synthesis of Ras(1-166)Gln61GluOMe. Refolding of the semisynthetic product in the presence of GTP was successful and revealed intrinsic GTPase activity of Ras(1-166)Gln61GluOMe.

UI MeSH Term Description Entries
D008958 Models, Molecular Models used experimentally or theoretically to study molecular shape, electronic properties, or interactions; includes analogous molecules, computer-generated graphics, and mechanical structures. Molecular Models,Model, Molecular,Molecular Model
D009154 Mutation Any detectable and heritable change in the genetic material that causes a change in the GENOTYPE and which is transmitted to daughter cells and to succeeding generations. Mutations
D010455 Peptides Members of the class of compounds composed of AMINO ACIDS joined together by peptide bonds between adjacent amino acids into linear, branched or cyclical structures. OLIGOPEPTIDES are composed of approximately 2-12 amino acids. Polypeptides are composed of approximately 13 or more amino acids. PROTEINS are considered to be larger versions of peptides that can form into complex structures such as ENZYMES and RECEPTORS. Peptide,Polypeptide,Polypeptides
D002384 Catalysis The facilitation of a chemical reaction by material (catalyst) that is not consumed by the reaction. Catalyses
D004926 Escherichia coli A species of gram-negative, facultatively anaerobic, rod-shaped bacteria (GRAM-NEGATIVE FACULTATIVELY ANAEROBIC RODS) commonly found in the lower part of the intestine of warm-blooded animals. It is usually nonpathogenic, but some strains are known to produce DIARRHEA and pyogenic infections. Pathogenic strains (virotypes) are classified by their specific pathogenic mechanisms such as toxins (ENTEROTOXIGENIC ESCHERICHIA COLI), etc. Alkalescens-Dispar Group,Bacillus coli,Bacterium coli,Bacterium coli commune,Diffusely Adherent Escherichia coli,E coli,EAggEC,Enteroaggregative Escherichia coli,Enterococcus coli,Diffusely Adherent E. coli,Enteroaggregative E. coli,Enteroinvasive E. coli,Enteroinvasive Escherichia coli
D004952 Esters Compounds derived from organic or inorganic acids in which at least one hydroxyl group is replaced by an –O-alkyl or another organic group. They can be represented by the structure formula RCOOR’ and are usually formed by the reaction between an acid and an alcohol with elimination of water. Ester
D006160 Guanosine Triphosphate Guanosine 5'-(tetrahydrogen triphosphate). A guanine nucleotide containing three phosphate groups esterified to the sugar moiety. GTP,Triphosphate, Guanosine
D006860 Hydrogen Bonding A low-energy attractive force between hydrogen and another element. It plays a major role in determining the properties of water, proteins, and other compounds. Hydrogen Bonds,Bond, Hydrogen,Hydrogen Bond
D006868 Hydrolysis The process of cleaving a chemical compound by the addition of a molecule of water.
D017510 Protein Folding Processes involved in the formation of TERTIARY PROTEIN STRUCTURE. Protein Folding, Globular,Folding, Globular Protein,Folding, Protein,Foldings, Globular Protein,Foldings, Protein,Globular Protein Folding,Globular Protein Foldings,Protein Foldings,Protein Foldings, Globular

Related Publications

Jelena Milić, and Ralf Seidel, and Christian F W Becker, and Roger S Goody, and Martin Engelhard
Protein engineering of cytochrome c by semisynthesis: substitutions at glutamic acid 66.
January 1986, Protein engineering,
Jelena Milić, and Ralf Seidel, and Christian F W Becker, and Roger S Goody, and Martin Engelhard
Detection of transforming ras proteins containing leucine at position 61 by a new mouse monoclonal antibody, ras(53-69)Leu61.
November 1989, Cancer research,
Jelena Milić, and Ralf Seidel, and Christian F W Becker, and Roger S Goody, and Martin Engelhard
H-ras mutations at codon 61 or 13 in tumors initiated with a NO donor in mouse skin.
June 2003, Cancer letters,
Jelena Milić, and Ralf Seidel, and Christian F W Becker, and Roger S Goody, and Martin Engelhard
A PCR-RFLP method for the detection of activated H-ras oncogene with a point mutation at codon 12 and 61.
December 1996, Yonsei medical journal,
Jelena Milić, and Ralf Seidel, and Christian F W Becker, and Roger S Goody, and Martin Engelhard
Enzymatic semisynthesis of insulin specifically labelled with tritium at position B-30.
January 1987, Protein engineering,
Jelena Milić, and Ralf Seidel, and Christian F W Becker, and Roger S Goody, and Martin Engelhard
The dominant negative effects of H-Ras harboring a Gly to Ala mutation at position 60.
November 1996, The Journal of biological chemistry,
Jelena Milić, and Ralf Seidel, and Christian F W Becker, and Roger S Goody, and Martin Engelhard
Structure-activity relationship of daptomycin analogues with substitution at (2S, 3R) 3-methyl glutamic acid position.
February 2017, Bioorganic & medicinal chemistry letters,
Jelena Milić, and Ralf Seidel, and Christian F W Becker, and Roger S Goody, and Martin Engelhard
Cleavage at glutamic acid with staphylococcal protease.
January 1977, Methods in enzymology,
Jelena Milić, and Ralf Seidel, and Christian F W Becker, and Roger S Goody, and Martin Engelhard
Quantitative Measurement of Intrinsic GTP Hydrolysis for Carcinogenic Glutamine 61 Mutants in H-Ras.
November 2018, Biochemistry,
Jelena Milić, and Ralf Seidel, and Christian F W Becker, and Roger S Goody, and Martin Engelhard
Mutations in residue 61 of H-Ras p21 protein influence MHC class II presentation.
February 1995, International immunology,
Copied contents to your clipboard!