Sequence-specific 1H and 15N resonance assignments and secondary structure of GDP-bound human c-Ha-Ras protein in solution |
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Authors: | Yutaka Muto Kazuhiko Yamasaki Yutaka Ito Shunsuke Yajima Haruhiko Masaki Takeshi Uozumi Markus Wälchli Susumu Nishimura Tatsuo Miyazawa Shigeyuki Yokoyama |
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Institution: | (1) Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, Hongo, Bunkyo-ku, 113 Tokyo, Japan;(2) Department of Biotechnology, Faculty of Agriculture, University of Tokyo, Hongo, Bunkyo-ku, 113 Tokyo, Japan;(3) Bruker Japan, Ninomiya, 305 Tsukuba, Ibaraki, Japan;(4) National Cancer Center Research Institute, Tsukiji, Chuo-ku, 104 Tokyo, Japan;(5) Protein Engineering Research Institute, Furuedai, 565 Suita, Osaka, Japan;(6) Present address: Banyu Tsukuba Research Institute in Collaboration with Merck Sharp and Dohme Research Laboratories, Banyu Pharmaceutical Co. Ltd., Tsukuba Techno-Park Oho, Okubo 3, 300-33 Tsukuba, Japan |
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Abstract: | Summary All the backbone 1H and 15N magnetic resonances (except for Pro residues) of the GDP-bound form of a truncated human c-Ha-ras proto-oncogene product (171 amino acid residues, the Ras protein) were assigned by 15N-edited two-dimensional NMR experiments on selectively 15N-labeled Ras proteins in combination with three-dimensional NMR experiments on the uniformly 15N-labeled protein. The sequence-specific assignments were made on the basis of the nuclear Overhauser effect (NOE) connectivities of amide protons with preceding amide and/or Cprotons. In addition to sequential NOEs, vicinal spin coupling constants for amide protons and C protons and deuterium exchange rates of amide protons were used to characterize the secondary structure of the GDP-bound Ras protein; six strands and five helices were identified and the topology of these elements was determined. The secondary structure of the Ras protein in solution was mainly consistent with that in crystal as determined by X-ray analyses. The deuterium exchange rates of amide protons were examined to elucidate the dynamic properties of the secondary structure elements of the Ras protein in solution. In solution, the -sheet structure in the Ras protein is rigid, while the second helix (A66-R73) is much more flexible, and the first and fifth helices (S17-124 and V152-L171) are more rigid than other helices. Secondary structure elements at or near the ends of the effector-region loop were found to be much more flexible in solution than in the crystalline state. |
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Keywords: | GTP binding protein Oncogene product Multi-dimensional NMR spectroscopy Stable isotope labeling |
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