Utilization of paramagnetic relaxation enhancements for structural analysis of actin-binding proteins in complex with actin.
Utilization of paramagnetic relaxation enhancements for structural analysis of actin-binding proteins in complex with actin.
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DOI：
10.1038/srep33690
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发表时间：
2016-09-22
影响因子：
4.6
通讯作者：
Shimada I
中科院分区：
文献类型：
--
作者：
Huang S;Umemoto R;Tamura Y;Kofuku Y;Uyeda TQ;Nishida N;Shimada I
肌动蛋白细胞骨架的动力学是由各种肌动蛋白结合蛋白（ABP）调节单体G-肌动蛋白的聚合和丝状F-肌动蛋白的解聚。虽然揭示的肌动蛋白/ABP复合物的结构是至关重要的了解如何ABPs调节肌动蛋白动力学，X射线晶体学和cryoEM方法是不足以适用于与G-或F-肌动蛋白的低亲和力或多种结合模式的ABPs。在这项研究中，我们的目的是建立替代的方法来建立一个结构模型的G-肌动蛋白/ABP复合物，利用顺磁弛豫增强（PRE）实验。胸腺素β4（Thymosin β 4，Tβ4）与G-肌动蛋白复合物的结构最近被报道，因此Tβ4被用作验证的测试用例。在特定位点用硝酰基自旋标记物标记含有半胱氨酸突变的G-肌动蛋白。根据T β4与G-actin复合物在顺磁性和反磁性状态下的1H-15 N HSQC谱的强度比，估算了Tβ4的酰胺基与G-actin自旋标记之间的距离.使用PRE导出的距离约束，我们能够计算出G-actin/Tβ4复合物的良好收敛的对接结构，该结构与参考结构非常一致。
Actin cytoskeleton dynamics are controlled by various actin binding proteins (ABPs) that modulate the polymerization of the monomeric G-actin and the depolymerization of filamentous F-actin. Although revealing the structures of the actin/ABP complexes is crucial to understand how the ABPs regulate actin dynamics, the X-ray crystallography and cryoEM methods are inadequate to apply for the ABPs that interact with G- or F-actin with lower affinity or multiple binding modes. In this study, we aimed to establish the alternative method to build a structural model of G-actin/ABP complexes, utilizing the paramagnetic relaxation enhancement (PRE) experiments. Thymosin β4 (Tβ4) was used as a test case for validation, since its structure in complex with G-actin was reported recently. Recombinantly expressed G-actin, containing a cysteine mutation, was conjugated with a nitroxyl spin label at the specific site. Based on the intensity ratio of the 1H-15N HSQC spectra of Tβ4 in the complex with G-actin in the paramagnetic and diamagnetic states, the distances between the amide groups of Tβ4 and the spin label of G-actin were estimated. Using the PRE-derived distance constraints, we were able to compute a well-converged docking structure of the G-actin/Tβ4 complex that shows great accordance with the reference structure.
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影响因子：
15
作者：
Nanga, Ravi P. R.;Brender, Jeffrey R.;Vivekanandan, Subramanian;Popovych, Nataliya;Ramamoorthy, Ayyalusamy
通讯作者：
Ramamoorthy, Ayyalusamy
DOI：
10.1016/s0006-291x(03)01133-1
发表时间：
2003-07-18
影响因子：
3.1
作者：
Akkari, PA;Nowak, KJ;Laing, NG
通讯作者：
Laing, NG
DOI：
10.1002/prot.340230306
发表时间：
1995-11-01
期刊：
PROTEINS-STRUCTURE FUNCTION AND GENETICS
影响因子：
--
作者：
SALI, A;POTTERTON, L;KARPLUS, M
通讯作者：
KARPLUS, M
影响因子：
64.8
作者：
Fujii, Takashi;Iwane, Atsuko H.;Namba, Keiichi
通讯作者：
Namba, Keiichi
影响因子：
11.4
作者：
Irobi, E;Aguda, AH;Robinson, RC
通讯作者：
Robinson, RC