Protein structure determination using long-distance constraints from double-quantum coherence ESR: Study of T4 lysozyme
Protein structure determination using long-distance constraints from double-quantum coherence ESR: Study of T4 lysozyme
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DOI：
10.1021/ja020040y
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发表时间：
2002-05-15
影响因子：
15
通讯作者：
Freed, JH
中科院分区：
文献类型：
--
作者：
Borbat, PP;Mchaourab, HS;Freed, JH
我们报告了一种基于双量子相干（DQC）检测的新型脉冲ESR距离测量技术的使用，该技术产生高质量的偶极光谱，使用氮氧化物自旋标记显着扩展了蛋白质可测量距离的范围。用DQCESR在9和17 GHz波段对8个T4溶菌酶（T4L）突变体进行了双甲基乙硫磺酸自旋标记（MTSSL）研究。距离从65/76突变体的20埃到61/135突变体的47埃不等。高质量的偶极光谱还允许确定距离分布，其宽度可用于在未来的计算策略中设置上界和下界。研究还表明，这些分布的形状可以揭示自旋标签的多种构象的存在，这是一个与距离的结构解释至关重要的问题。根据已知的晶体结构、氮氧化物侧链的特征构象和分子模型，本研究中发现的距离和分布很容易合理化。这项研究为使用DQC-ESR来确定大蛋白的三级结构奠定了基础，只有少量的长距离限制。
We report the use of a novel pulsed ESR technique for distance measurement, based on the detection of double quantum coherence (DQC), which yields high quality dipolar spectra, to significantly extend the range of measurable distances in proteins using nitroxide spin-labels. Eight T4 lysozyme (T4L) mutants, doubly labeled with methanethiosulfonate spin-label (MTSSL), have been studied using DQCESR at 9 and 17 GHz. The distances span the range from 20 Angstrom for the 65/76 mutant to 47 Angstrom for the 61/135 mutant. The high quality of the dipolar spectra also allows the determination of the distance distributions, the width of which can be used to set upper and lower bounds in future computational strategy. It is also demonstrated that the shape of these distributions can reveal the presence of multiple conformations of the spin-label, an issue of critical relevance to the structural interpretation of the distances, The distances and distributions found in this study are readily rationalized in terms of the known crystal structure, the characteristic conformers of the nitroxide side chains, and molecular modeling, This study sets the stage for the use of DQC-ESR for determining the tertiary structure of large proteins with just a small number of long-distance constraints.