EPR和NMR自旋标记法研究部分折叠蛋白质构象

摘要/Abstract

摘要：

部分折叠蛋白质的特征是在溶液中有部分二级结构，但是三级结构接触比较松散或者较少，其意义可能是蛋白质折叠过程的中间态，或者有重要的生理功能. 利用EPR和NMR波谱可以表征其构象特征，从而构建其二级结构、三级结构、四级结构以及构象变化的结构模型. 利用分子生物学的点突变技术可以在蛋白质主链上插入1个或2个半胱氨酸残基，然后把顺磁自旋标记物特异地共价结合在半胱氨酸的侧链巯基上来制备自旋标记样品. 位点特异性自旋标记电子顺磁共振（SDSL-EPR）波谱是通过测定2个自旋标记间的偶极相互作用，从而推测2个硝基氧自由基间的距离. 核磁共振（NMR）波谱则是通过测定单个自旋标记中心对周围原子核驰豫效应增强（PRE）的效应，推测出顺磁中心相对于周围原子核的距离. 连续波EPR和NMR自旋标记方法可以测定2.5 nm左右的偶极相互作用距离，属于长程的距离约束，这对于确定部分折叠蛋白质的构象至关重要. 该文将就蛋白质部分折叠态研究的生物学意义，自旋标记方法以及EPR和NMR方法研究其构象特征举例描述.

关键词: 电子顺磁共振（EPR）, 核磁共振（NMR）, 部分折叠蛋白质, 构象, 距离约束

Abstract:

Partially folded proteins, characterized as exhibiting secondary structure elements with loose or absent tertiary contacts, are important intermediates in physiological protein folding and under pathological conditions. Structural restraints derived from EPR and NMR spectroscopy can be used to test and build secondary, tertiary, and quaternary structural models as well as measure protein conformational changes in solution. Insertion of one or two cysteine residues into the protein backbone using molecular biology methods and the subsequent labeling of the cysteine residues with a paramagnetic spin label enables the techniques of both EPR and NMR to be used as the molecular spectroscopic ruler. Site-directed spin labeling electron paramagnetic resonance (SDSL-EPR) measures the dipolar interaction between pairs of paramagnetic spin labels to yield inter-nitroxide distances, while the method of paramagnetic relaxation enhancement (PRE) measured by NMR gives information on the distance between the spin labeling center and the nuclei. The inter-spin dipolar interactions at about 2.5 nm are measured using continuous wave (CW) EPR and NMR methods. As for any low-resolution distance methods, the positioning of the spin labels and the number of distance constraints to be measured are depends on the structural question being asked, thus a pattern approach for using distance sets to decipher structure mapping, including protein folds and conformational changes associated with biological activity, is essential.

Key words: EPR, NMR, partially folded protein, conformation, distance restraints

中图分类号:

O482.53

朱磊, 刘志军. EPR和NMR自旋标记法研究部分折叠蛋白质构象[J]. 波谱学杂志, 2011, 28(4): 523-533.

ZHU Lei, LIU Zhi-Jun. Conformational Studies of Partially Folded Protein by EPR and NMR Spin Labeling Methods: A Review[J]. Chinese Journal of Magnetic Resonance, 2011, 28(4): 523-533.

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