Semisynthesis of H-Ras with a glutamic acid methylester at position 61.
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.
Milić
Jelena
J
Max-Planck-Institut für Molekulare Physiologie, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany.
Seidel
Ralf
R
Becker
Christian F W
CF
Goody
Roger S
RS
Engelhard
Martin
M
D002384:Catalysis; D004926:Escherichia coli / Q000235:genetics; D004952:Esters / Q000737:chemistry*; D020558:GTP Phosphohydrolases / Q000378:metabolism; D018698:Glutamic Acid / Q000737:chemistry*; D006160:Guanosine Triphosphate / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006860:Hydrogen Bonding; D006868:Hydrolysis; D008958:Models, Molecular; D009154:Mutation; D010455:Peptides / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism; D017510:Protein Folding; D018631:ras Proteins / Q000138:chemical synthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D004952:Esters; D010455:Peptides; D018698:Glutamic Acid; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases; D018631:ras Proteins