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1.
Biochemistry ; 35(37): 12186-93, 1996 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-8810926

RESUMO

As a molecular switch, the ras protein p21 undergoes structural changes that couple recognition sites on the protein surface to the guanine nucleotide-divalent metal ion binding site. X-ray crystallographic studies of p21 suggest that 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 more distant from the metal and were interpreted as weak or indirect coordination of this residue. We report high frequency (139.5 GHz) EPR spectroscopy of p21.Mn(II) complexes of two guanine nucleotides that probes the link between threonine-35 and the divalent metal ion. By analysis of high-frequency EPR spectra, we determine the number of water molecules in the first coordination sphere of the manganous ion to be four in p21.Mn(II).GDP, consistent with prior low-frequency EPR and X-ray crystallographic studies. In the complex of p21 with a GTP analog, p21.Mn(II).GMPPNP, we determine the hydration number to be 2, also consistent with crystal structures. This result rules out indirect coordination of threonine-35 in the solution structure of p21.Mn(II).GMPPNP, and implicates direct, weak coordination of this residue as suggested by Halkides et al. [(1994) Biochemistry 33,4019]. The 17O hyperfine coupling constant of H2(17)O is determined as 0.25 mT in the GDP from and 0.28 mT in the GTP form. These values are similar to reported values for 17O-enriched aquo ligands and some phosphato ligands in Mn(II) complexes. The high magnetic field strength (4.9 T) employed in these 139.5 GHz EPR measurements leads to a narrowing of the Mn(II) EPR lines that facilitates the determination of 17O hyperfine interactions.


Assuntos
Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/química , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Guanilil Imidodifosfato/metabolismo , Manganês , Modelos Químicos , Isótopos de Oxigênio , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Treonina , Água
2.
Biochemistry ; 35(37): 12194-2200, 1996 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-8810927

RESUMO

Electron paramagnetic resonance spectroscopy at 139.5 GHz has been used to study p21 ras complexed with Mn(II) and guanosine 5'-(beta, gamma-imidotriphosphate), an analog of GTP. The p21 sample studied was selectively labeled with [17O gamma]threonine to a final enrichment of 30%. A Mn(II)-17O hyperfine interaction was observed, but the value of the coupling constant, 0.11 +/- 0.04 mT, is the smallest such value yet reported. Ab initio calculations indicate that this value is consistent with direct coordination of the threonine hydroxyl group and provide an estimate for the Mn(II)-17O bond length of 2.7 A. The measured hyperfine coupling constant and associated bond length starkly contrast with typical values for Mn(II)-17O coordination complexes, namely, approximately 0.25 mT and approximately 2.2 A, respectively. This contrast underscores the peculiar weakness of this Mn(II)-O interaction in p21 and persuasively argues that the nucleotide-induced conformational change, which is known to encompass the region of p21 involving Thr35, is not driven by Mn(II) coordination of the Thr35 hydroxyl group.


Assuntos
Guanosina Trifosfato/metabolismo , Conformação Proteica , Proteínas Proto-Oncogênicas p21(ras)/química , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Treonina , Sítios de Ligação , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Cromatografia Gasosa-Espectrometria de Massas , Guanilil Imidodifosfato/metabolismo , Cinética , Manganês/metabolismo , Isótopos de Oxigênio , Ligação Proteica
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