Structural and dynamic differences between normal and transforming N-ras gene products: a 31P and isotope-edited 1H NMR study.
[15N]Glycine was biosynthetically incorporated into normal cellular N-ras p21 and a position 12 transforming mutant, in order to produce p21 proteins containing several site-specific NMR probes at or near activating positions in the guanine nucleotide binding domain. We have previously assigned all five glycine resonances located in loops directly involved in binding of guanosine diphosphate in the wild-type p21 protein [Campbell-Burk, S., Papastavros, M. Z., McCormick, F., & Redfield, A. G. (1989) Proc. Natl. Acad. Sci. U.S.A 86, 817-820]. In this report, the corresponding glycine resonances in the p21 mutant have been assigned, and spectral differences between normal and mutant p21-guanosine diphosphate (p21.GDP) complexes have been investigated. Our combined 1H[15N] and 31P NMR results show that substitution of aspartate for glycine-12 produces perturbations in the phosphoryl binding domain, near the point of the mutation. Although many of the remaining glycines were unaffected, spectral differences were also observed outside the GDP binding domain. Two of the five active-site glycines in wild-type p21.GDP have very slow amide proton exchange rates with water (kappa less than 2.8 x 10(-5) s-1). The active-site glycines are located in solvent-exposed loops, so their apparent solvent inaccessibility may result from strong hydrogen bond formation between glycine amide protons and bound guanine diphosphate and/or other nearby groups in p21.
Campbell-Burk
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Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02254.
D001224:Aspartic Acid; D001665:Binding Sites; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D005998:Glycine; D006153:Guanosine Diphosphate / Q000378:metabolism; D006860:Hydrogen Bonding; D009682:Magnetic Resonance Spectroscopy; D009154:Mutation; D011518:Proto-Oncogene Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D016283:Proto-Oncogene Proteins p21(ras); D013057:Spectrum Analysis
D011518:Proto-Oncogene Proteins; D006153:Guanosine Diphosphate; D001224:Aspartic Acid; D016283:Proto-Oncogene Proteins p21(ras); D005998:Glycine