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首页> 外文期刊>Physical chemistry chemical physics: PCCP >Conserved electrostatic fields at the Ras-effector interface measured through vibrational Stark effect spectroscopy explain the difference in tilt angle in the Ras binding domains of Raf and RalGDS
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Conserved electrostatic fields at the Ras-effector interface measured through vibrational Stark effect spectroscopy explain the difference in tilt angle in the Ras binding domains of Raf and RalGDS

机译：通过振动斯塔克效应光谱测量的Ras效应子界面上的守恒静电场解释了Raf和RalGDS的Ras结合域中倾斜角的差异

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Vibrational Stark effect (VSE) spectroscopy was used to measure the electrostatic fields present at the interface of the human guanosine triphosphatase (GTPase) Ras docked with the Ras binding domain (RBD) of the protein kinase Raf. Nine amino acids located on the surface of Raf were selected for labeling with a nitrile vibrational probe. Eight of the probe locations were situated along the interface of Ras and Raf, and one probe was 2 nm away on the opposite side of Raf. Vibrational frequencies of the nine Raf nitrile probes were compared both in the monomeric, solvated protein and when docked with wild-type (WT) Ras to construct a comprehensive VSE map of the Ras-Raf interface. Molecular dynamics (MD) simulations employing an umbrella sampling strategy were used to generate a Boltzmann-weighted ensemble of nitrile positions in both the monomeric and docked complexes to determine the effect that docking has on probe location and orientation and to aid in the interpretation of VSE results. These results were compared to an identical study that was previously conducted on nine nitrile probes on the RBD of Ral guanidine dissociation stimulator (RalGDS) to make comparisons between the docked complexes formed when either of the two effectors bind to WT Ras. This comparison finds that there are three regions of conserved electrostatic fields that are formed upon docking of WT Ras with both downstream effectors. Conservation of this pattern in the docked complex then results in different binding orientations observed in otherwise structurally similar proteins. This work supports an electrostatic cause of the known binding tilt angle between the Ras-Raf and Ras-RalGDS complexes.
机译：振动斯塔克效应（VSE）光谱用于测量与蛋白激酶Raf的Ras结合结构域（RBD）相连的人鸟苷三磷酸酶（GTPase）Ras界面上存在的静电场。选择位于Raf表面的9个氨基酸，用腈振动探针进行标记。八个探针位置沿Ras和Raf的界面放置，一个探针在Raf的另一侧相距2 nm。比较了九种Raf腈探针在单体溶剂化蛋白质中以及与野生型（WT）Ras对接时的振动频率，以构建Ras-Raf界面的全面VSE图。使用伞式采样策略的分子动力学（MD）模拟用于在单体和对接复合物中生成腈原子的玻尔兹曼加权集合，以确定对接对探针位置和方向的影响，并有助于解释VSE结果。将这些结果与先前在Ral胍离解刺激物（RalGDS）的RBD上的九个腈探针上进行的一项相同研究进行了比较，以比较两种效应子之一与WT Ras结合时形成的对接复合物。该比较发现，在WT Ras与两个下游效应子对接时形成了三个守恒静电场区域。然后，在对接的复合物中保守该模式会导致在其他结构相似的蛋白质中观察到不同的结合方向。这项工作支持了Ras-Raf和Ras-RalGDS配合物之间已知结合倾斜角的静电原因。

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