Accurate long-range distance measurements in a doubly spin-labeled protein by a four-pulse, double electron-electron resonance method
Accurate long-range distance measurements in a doubly spin-labeled protein by a four-pulse, double electron-electron resonance method
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DOI：
10.1002/mrc.2290
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发表时间：
2008-12-01
影响因子：
2
通讯作者：
Huber, Martina
中科院分区：
文献类型：
--
作者：
Finiguerra, Michela G.;Prudencio, Miguel;Huber, Martina
用四脉冲双电子-电子共振法(DEER或PELDOR)测量无序系统中的距离，因为可以测量很长的距离(几个纳米)及其分布，所以变得越来越受欢迎。从距离分布可以推断出最终的非均质性和动力学。为了充分利用该方法，需要对结构明确的系统进行典型的距离分布。在这里，通过定点突变的方法连接两个(1-氧基-2，2，5，5-四甲基-2，2，5，5-四甲基吡咯烷-3-甲基)甲硫磺酸自旋标记(MTSL)来研究结构特征良好的天青蛋白。在双突变体Q12C/K27C和K27C/N42C中，蛋白Q12、K27和N42的表面位点发生突变。Q12C/K27C的距离为4.3 nm，K27C/N42C的距离为4.6 nm。Q12C/K27C的分布宽度(0.24 nm)小于K27C/N42C突变体的分布宽度(0.36 nm)。分布的形状接近于高斯分布。这些距离分布与用于确定自旋标记连接物的最大可达构象空间的模型的距离分布很好地吻合。此外，还模拟了较短距离变异Q12C/N42C的预期分布。Q12C/K27C的宽度比Q12C/K27C的计算值大21%，反映了不同取向和较短距离的影响。分布的宽度和形状适合作为在结构上定义良好的位置处的两个未受干扰的MTSL标签的参考。版权所有(C)2008 John Wiley&Sons，Ltd.
Distance determination in disordered systems by a four-pulse double electron-electron resonance method (DEER or PELDOR) is becoming increasingly popular because long distances (several nanometers) and their distributions can be measured. From the distance distributions eventual heterogeneities and dynamics can be deduced. To make full use of the method, typical distance distributions for structurally well-defined systems are needed. Here, the structurally well-characterized protein azurin is investigated by attaching two (1-oxyl-2,2,5,5-tetra met hylpyrroline-3-methyl) methanethiosulfonate spin labels (MTSL) by site-directed mutagenesis. Mutations at the surface sites of the protein Q12, K27, and N42 are combined in the double mutants Q12C/K27C and K27C/N42C. A distance of 4.3 nm is found for Q12C/K27C and 4.6 nm for K27C/N42C. For Q12C/K27C the width of the distribution (0.24 nm) is smaller than for the K27C/N42C mutant (0.36 nm). The shapes of the distributions are close to Gaussian. These distance distributions agree well with those derived from a model to determine the maximally accessible conformational space of the spin-label linker. Additionally, the expected distribution for the shorter distance variant Q12C/N42C was modeled. The width is larger than the calculated one for Q12C/K27C by 21%, revealing the effect of the different orientation and shorter distance. The widths and the shapes of the distributions are suited as a reference for two unperturbed MTSL labels at structurally well-defined sites. Copyright (C) 2008 John Wiley & Sons, Ltd.