HIGH FIELD EPR OF MN 21 P2L RAS

作为一个分子开关，ras蛋白经历了结构变化。 将蛋白质表面的识别位点连接到 鸟嘌呤核苷酸-二价金属离子结合部位。X射线 P21的结晶学研究表明，p21与 苏氨酸-35和二价金属离子在 这些构象变化。P21基因在ESEEM中的最新研究 然而，解决方案是将苏氨酸-35放在更远离金属的地方，并 被解释为该残基的弱配位或间接配位。我们 报道了p21*Mn(II)的高频(139.5 GHz)电子顺磁共振谱 两个交替的鸟嘌呤核苷酸的络合物探测连接 在苏氨酸-35和二价金属离子之间。尤其是， 水分子在第一配位球中的数量 测定了P21-1-Mn-(Il)-GDP中的四个锰离子和两个 在p2I-Mn(H)-GMPPNP中。GMPPNP(一种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，并与这一直接的，弱的配位一致 哈尔基德斯建议的残留物。170超精细耦合常数 在国内生产总值形式中确定为0.25公吨，在 GTP表。这些值与报告的 其他锰(II)络合物中的170个富含水和磷的配体。 对于所有这些测量，高磁场强度(4.9 T)，对应于139.5 GHz EPR激发，产生窄的Mn(II) 从而提高对170超精细展宽的敏感度。

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.