Abstract

As a molecular switch the ras protein undergoes structural changes that couple recognition sites on the surface of the protein to the guanine nucleotide-divalent metal ion binding site. X-ray crystallographic studies of p21 suggest coordination between threonine-35 and the divalent metal ion plays an important role in these conformational changes. Recent ESEEM studies of p21 in solution, however, place threonine-35 further away from the metal and were interpreted as weak or indirect coordination of this residue. We report high frequency (139.5 GHz) pulsed EPR spectroscopy of p21?Mn(II) complexes of GMPPNP that probe the link between threonine-35 and the divalent metal ion. In particular, we determine the 17O hyperfine coupling constant of [17Og]Thr 35 is determined to be ~1.1 G in the GTP form. This value is much smaller than reported values (1.6 - 4 G) for 17O-enriched aquo-and phosphato-ligands in other complexes of Mn(II). These results are in agreement with direct, weak coordination of this residue as suggested by Halkides et al. (1994) Biochemistry 33, 4019.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Biotechnology Resource Grants (P41)
Project #
5P41RR000995-21
Application #
5221917
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
21
Fiscal Year
1996
Total Cost
Indirect Cost

  Publications

Marintchev, Assen; Edmonds, Katherine A; Marintcheva, Boriana et al. (2009) Topology and regulation of the human eIF4A/4G/4H helicase complex in translation initiation. Cell 136:447-60
Frueh, Dominique P; Arthanari, Haribabu; Koglin, Alexander et al. (2009) A double TROSY hNCAnH experiment for efficient assignment of large and challenging proteins. J Am Chem Soc 131:12880-1
Frueh, Dominique P; Leed, Alison; Arthanari, Haribabu et al. (2009) Time-shared HSQC-NOESY for accurate distance constraints measured at high-field in (15)N-(13)C-ILV methyl labeled proteins. J Biomol NMR 45:311-8
Lentz, Margaret R; Westmoreland, Susan V; Lee, Vallent et al. (2008) Metabolic markers of neuronal injury correlate with SIV CNS disease severity and inoculum in the macaque model of neuroAIDS. Magn Reson Med 59:475-84
Chen, Jingyang; Dupradeau, Francois-Yves; Case, David A et al. (2007) Nuclear magnetic resonance structural studies and molecular modeling of duplex DNA containing normal and 4'-oxidized abasic sites. Biochemistry 46:3096-107
Hyberts, Sven G; Heffron, Gregory J; Tarragona, Nestor G et al. (2007) Ultrahigh-resolution (1)H-(13)C HSQC spectra of metabolite mixtures using nonlinear sampling and forward maximum entropy reconstruction. J Am Chem Soc 129:5108-16
Lentz, Margaret R; Kim, John P; Westmoreland, Susan V et al. (2005) Quantitative neuropathologic correlates of changes in ratio of N-acetylaspartate to creatine in macaque brain. Radiology 235:461-8
Kim, John P; Lentz, Margaret R; Westmoreland, Susan V et al. (2005) Relationships between astrogliosis and 1H MR spectroscopic measures of brain choline/creatine and myo-inositol/creatine in a primate model. AJNR Am J Neuroradiol 26:752-9
Peled, S; Cory, D G; Raymond, S A et al. (1999) Water diffusion, T(2), and compartmentation in frog sciatic nerve. Magn Reson Med 42:911-8
Mo, H; Dai, Y; Pochapsky, S S et al. (1999) 1H, 13C and 15N NMR assignments for a carbon monoxide generating metalloenzyme from Klebsiella pneumoniae. J Biomol NMR 14:287-8

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