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

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Abstract
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-51 or Cys-80, was found to be 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 acontrol 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. Thus, the MSL group was found to be in the vicinity of the protein-bound GDP. A phenylalanine residue and two histidine residues, which were characterized by 2D HOHAHA and DOF-COSY spectra, were also found to be in the vicinity of MSL. 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 [de Vox, A. M., Tong. L., Milburn, M. V., Matias, P. M., Jancarik, J., Noguchi, S., Nishimura, S., Miura, K., Ohtsuka, E., and Kim, S.-H. (1988) Science 239, 888-893], 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.