HIGH FIELD EPR OF MN 21 P2L RAS

ras蛋白作为一种分子开关， 将蛋白质表面的识别位点 鸟嘌呤核苷酸-二价金属离子结合位点。 x射线 P2 L的晶体学研究表明， 苏氨酸-35和二价金属离子起着重要的作用， 这些构象变化。 最近的ESEEM研究p2 l在 然而，溶液将苏氨酸-35放置得更远离金属， 被解释为弱或间接协调的这一残余物。 我们 报道了p2 l *Mn（II）的高频（139.5GHz）EPR谱 两个交替的鸟嘌呤核苷酸的复合物， 在苏氨酸-35和二价金属离子之间。 特别是 第一配位层中的水分子数 的 在p_2_1-Mn-（II）-GDP中测得四价锰离子， 在p2 I-Mn（H）-GMPPNP中。 GMPPNP（GTP类似物）的结果如下： 与X射线预测的水分子数量一致 结构 这些结果排除了间接协调 苏氨酸-35，并与直接，弱协调一致，这 残基，如Halkides所建议的。 170超精细耦合常数 H2 170在GDP形式中被确定为0.25 mT，在 GTP形式。 这些值与报告的 170-在其他Mn（II）络合物中富集了水和磷酸根配体。 对于所有这些测量，高磁场强度（4.9 T），对应于139.5GHz EPR激发，产生窄Mn（II） 线宽，从而提高了对170超精细加宽的灵敏度。

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 p2l suggest that coordination between threonine-35 and the divalent metal ion plays an important role in these conformational changes. Recent ESEEM studies of p2l in solution, however, place threonine-35 further away from the metal and were interpreted as weak or indirect coordination of this residue. We have reported the high frequency (139.5 GHz) EPR spectra of p2l*Mn(II) complexes of two alternate guanine nucleotides that probe the link between threonine-35 and the divalent metal ion. In particular, the number of water molecules in the first coordination sphere of the manganous ion was determined to be four in p2 1 -Mn-(Il)-GDP and two in p2 I -Mn(H)-GMPPNP. The results for GMPPNP (a GTP-analog) are consistent with the number of water molecules predicted by the X-ray structure. These results rule out indirect coordination of threonine-35 and are consistent with direct, weak coordination of this residue as suggested by Halkides. The 170 hyperfine coupling constant of H2 170 is determined as 0.25 mT in the GDP form and 0.28 mT in the GTP form. These values are similar to reported values for 170-enriched aquo- and phosphato-ligands in other complexes of Mn(II). For all of these measurements, the high magnetic field strength (4.9 T), corresponding to 139.5 GHz EPR excitation, yields narrow Mn(II) linewidths and thus enhances sensitivity to 170 hyperfine broadening.