Characterization of the active site of p21 ras by electron spin-echo envelope modulation spectroscopy with selective labeling: comparisons between GDP and GTP forms.
Characterization of the active site of p21 ras by electron spin-echo envelope modulation spectroscopy with selective labeling: comparisons between GDP and GTP forms.
复制标题
通过选择性标记的电子自旋回波包络调制光谱表征 p21 ras 的活性位点：GDP 和 GTP 形式之间的比较。
DOI：
10.1021/bi00179a031
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发表时间：
1994
期刊：
影响因子：
2.9
通讯作者：
Singel,DJ
中科院分区：
文献类型：
--
作者：
Halkides,CJ;Farrar,CT;Larsen,RG;Redfield,AG;Singel,DJ
1994年1月24日接收的修订版Mandalpt ®摘要：通过电子自旋回波包络调制光谱研究了连接到MnnGDP或MnnGMPPNP的人H-或N-rar p21的选择性标记样品，以确定二价金属周围的蛋白质环境。我们将[4-13* C]标记的Asx掺入p21-MnnGDP中，发现Asp 57的羧基13 C与Mn 11的距离约为4.1 nm。我们的结果与该残基与金属的间接配位一致。从[2- 2 H] Thr标记的样品中，我们估计从Mn 11离子到Thr 35的2 H的距离至少为5.8 nm。因此，金属的唯一蛋白质或核苷酸配体似乎是Ser 17和GDP的3-磷酸，如先前报道的[Larsen，RG，Halkides，C. J.，Redfield，A. G.，& Singel，D. J.等人（1992 b）J. Am.在p21的S ′-鸟苷酰亚胺二磷酸（GMPPNP）形式中，X射线晶体学已经报道Thr 35通过其羟基是金属的配体，并且该残基似乎在核苷酸取代后的生物学上重要的构象变化中起关键作用[派，E. F.、克伦格尔大学，Petsko，G.，古迪河美国，Kabsch，W.，& Wittinghousehold，A.（1990）EMBO J.9，2351-2359]。用[2- 2 H] Thr标记的p21·的ESEEM光谱产生4.9 μ m的Mnn*-2H距离，该距离与强配位不一致。其中Thr残基被13 C和15 N完全标记的p21样品产生的Mn 11到Thr 35的酰胺氮的距离值为5.0 μ m，而如果Thr 35配位，则13 C信号远小于预期。[15 N]丝氨酸/甘氨酸标记的样品与Ser 17的酰胺15 N之间的距离为3.9 nm，与X射线结构一致;[4- 13 C]标记的p21 Asx样品在Mn 11和Asp 57标记之间的距离约为4 nm，再次表明间接配位。这两个值都是
Revised Manuscript Received January 24, 1994® abstract: Selectively labeled samples of human H-or N-rar p21 ligated to MnnGDP or MnnGMPPNP were studied by electron spin-echo envelope modulation spectroscopy in order to define the protein environment around the divalent metal. We incorporated [4-13* C]-labeled Asx into p21-MnnGDP and found that the distance from the carboxyl13C of Asp57 to Mn11 is~4.1 Á. Our result is consistent with indirect coordination of this residue to the metal. From a [2-2H] Thr-labeled sample, we estimate that the distance from the Mn11 ion to the 2H of Thr35 is at least 5.8 Á. Thus, the only protein or nucleotide ligands to the metal appear to be Ser 17 and the 3-phosphate of GDP, as previously reported [Larsen, RG, Halkides, C. J., Redfield, A. G., & Singel, D. J.(1992b) J. Am. Chem. Soc. 114, 9608-9611], In the S'-guanylylimido diphosphate (GMPPNP) form of p21, Thr35 has been reported by X-ray crystallography to be a ligand of the metal via its hydroxyl group, and this residue appears to play a key role in the biologicallyimportant conformational change upon nucleotidesubstitution [Pai, E. F., Krengel, U., Petsko, G., Goody, R. S., Kabsch, W., & Wittinghofer, A.(1990) EMBO J. 9, 2351-2359]. The ESEEM spectrum of p21· labeled with [2-2H] Thr yields a Mnn*-2H distance of 4.9 Á, a distance inconsistent with strong coordination. A sample of p21 in which the Thr residues were fully labeled with 13C and 15N yielded a value of 5.0 Á for the distance from Mn11 to the amide nitrogen of Thr35, while the 13C signal is much smaller than expected ifThr35 were coordinated. A [15N] serine/glycine-labeled sample gives a distance to the amide 15N of Ser 17 of 3.9 Á, consistent with the X-ray structure; a [4-13C]-labeled Asx sample of p21 gives a distance of~ 4 Á between Mn11 and the label of Asp57, again implying indirect coordination. Both of these values are