University of Regensburg, Institute of Physical Biochemistry and Biophysics, Universitätsstrasse 31, D-93053 Regensburg, Germany.
J Am Chem Soc. 2011 Feb 23;133(7):2048-51. doi: 10.1021/ja108779j. Epub 2011 Jan 26.
(31)P NMR spectroscopy is a suitable method for identifying conformational states in the active site of guanine nucleotide binding proteins detecting the nucleotide placed there. Because there is no labeling necessary, this method is gaining increasing interest. By (31)P NMR spectroscopy two major conformational states, namely state 1(T) and state 2(T), can be detected in active Ras protein characterized by different chemical shifts. Depending on the conformational state Ras shows clearly different physiological properties. Meanwhile analogous conformational equilibria could also be shown for other members of the Ras superfamily. It is often difficult to determine the conformational states of the proteins on the basis of chemical shift alone; therefore, direct detection would be a great advantage. With the use of Cu(2+)-cyclen which selectively interacts only with one of the major conformational states (state 1) one has a probe to distinguish between the two states, because only proteins existing in conformational state 1 interact with the Cu(2+)-cyclen at low millimolar concentrations. The suitability was proven using Ras(wt) and Ras mutants, Ras complexed with GTP, GppNHp, or GTPγS, as well as two further members of the Ras superfamily namely Arf1 and Ran.
(31)P NMR 光谱学是一种适用于鉴定鸟嘌呤核苷酸结合蛋白活性部位构象状态的方法，可检测到该处的核苷酸。由于不需要标记，这种方法越来越受到关注。通过 (31)P NMR 光谱学，可以检测到活性 Ras 蛋白中的两种主要构象状态，即状态 1(T)和状态 2(T)，其特征是化学位移不同。根据构象状态，Ras 表现出明显不同的生理特性。同时，类似的构象平衡也可以在 Ras 超家族的其他成员中显示。仅基于化学位移很难确定蛋白质的构象状态；因此，直接检测将是一个很大的优势。使用仅与主要构象状态之一（状态 1）选择性相互作用的 Cu(2+)-环辛烷，我们有一个探针来区分这两种状态，因为只有处于构象状态 1 的蛋白质在低毫摩尔浓度下与 Cu(2+)-环辛烷相互作用。使用 Ras(wt)和 Ras 突变体、与 GTP、GppNHp 或 GTPγS 结合的 Ras、以及 Ras 超家族的另外两个成员 Arf1 和 Ran 证明了其适用性。
