Spin-labeling proton NMR study on aromatic amino acid residues in the guanine nucleotide binding site of human c-Ha-ras(1-171) protein

Journal Article · Tue Nov 28 00:00:00 EST 1989 · Biochemistry; (USA)

DOI:https://doi.org/10.1021/bi00450a045· OSTI ID:6854939

Hata-Tanaka, Akiko; Nishimura, Susumu ^[1]; Kawai, Gota; Yamasaki, Kazuhiko; Ito, Yutaka; Ha, Jongmyung; Yokoyama, Shigeyuki ^[2]; Kajiura, Hiroko ^[3]; Miyazawa, Tatsuo ^[4]

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1. National Cancer Center Research Institute, Tokyo (Japan)
2. Univ. of Tokyo (Japan)
3. National Institute for Basic Biology, Okazaki (Japan)
4. Yokohama National Univ. (Japan)

A truncated human c-Ha-ras gene product, ras(1-171) protein, was prepared and chemically modified with maleimide spin-label (MSL). By trypsin digestion of the MSL-labeled ras(1-171) protein, MSL-labeled peptide fragments were isolated and sequenced. The cysteine residue in position 118 of the protein, but not the other cysteine residues, Cys-51 or Cys-80, was found to specifically labeled by MSL. The ESR spectrum of the MSL-labeled ras(1-171) protein indicates that the MSL group attached to Cys-118 is strongly immobilized. Proton NMR spectra at 400-MHz were measured for this MSL-labeled ras(1-171) protein and also for a control sample of a labeled ras(1-171) protein whose MSL was reduced by sodium ascorbate. In the difference spectra for these two proteins, resonances of protons in the vicinity of the MSL group attached to Cys-118 of the ras(1-171) protein were observed. NOE and pH titration analyses indicate that this phenylalanine residue is close to the bound GDP and one of the two histidine residues. By carboxypeptidase digestion, the two histidine residues near MSL were identified as His-27 and His-94. On the basis of these observations and X-ray analysis it was concluded that Phe-28 and His-27 are in the proximity of GDP and MSL-labeled Cys-118. The role of the interaction between the aromatic ring of Phe-28 and the guanine ring of the bound GDP is discussed with regard to the mechanism of the ras protein functions.

• OSTI ID: 6854939
• Journal Information: Biochemistry; (USA), Vol. 28:24; ISSN 0006-2960
• Country of Publication: United States
• Language: English

Related Subjects

62 RADIOLOGY AND NUCLEAR MEDICINE
HISTIDINE
CHEMICAL SHIFT
MEMBRANE PROTEINS
NUCLEAR MAGNETIC RESONANCE
NUCLEOTIDES
CROSS-LINKING
AMINO ACIDS
AROMATICS
CYSTEINE
GUANINE
HEAVY WATER
ONCOGENES
PH VALUE
PHENYLALANINE
PROTONS
AMINES
AZAARENES
AZOLES
BARYONS
CARBOXYLIC ACIDS
CHEMICAL REACTIONS
ELEMENTARY PARTICLES
FERMIONS
GENES
HADRONS
HETEROCYCLIC ACIDS
HETEROCYCLIC COMPOUNDS
HYDROGEN COMPOUNDS
HYDROXY COMPOUNDS
IMIDAZOLES
MAGNETIC RESONANCE
NUCLEONS
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
ORGANIC SULFUR COMPOUNDS
OXYGEN COMPOUNDS
POLYMERIZATION
PROTEINS
PURINES
RESONANCE
THIOLS
WATER
550601* - Medicine- Unsealed Radionuclides in Diagnostics