Motion of spin-labeled side chains in T4 lysozyme:: Effect of side chain structure
Motion of spin-labeled side chains in T4 lysozyme:: Effect of side chain structure
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DOI：
10.1021/bi9826310
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发表时间：
1999-03-09
期刊：
影响因子：
2.9
通讯作者：
Hubbell, WL
中科院分区：
文献类型：
--
作者：
Mchaourab, HS;Kálai, T;Hubbell, WL
以前的研究表明，从电子顺磁共振(EPR)谱推断的蛋白质中氮氧基侧链的迁移率可以用来将特定的位置分类为螺旋表面位置、三级接触位置、埋藏位置或环状位置。此外，迁移率的序列依赖性可以识别规则的二级结构。然而，在最广泛使用的侧链中，氮氧基环与蛋白质在某些螺旋表面位置的明显相互作用导致EPR谱与三级接触点的EPR谱简并。在本研究中，我们使用T4溶菌酶中的特定位点来评估旨在解决这种简并问题的新型氮氧化物侧链。结果表明，试剂3-(methanesulfonylthiomethyl)-2，2，5，5-tetramethylpyrrolidin-1-yloxy与半胱氨酸反应生成了一个氮氧基侧链，该侧链在螺旋表面和三级接触点之间具有较高的迁移率，有效地解决了简并问题。试剂3-(iodomercuriomethyl)-2，2，5，5-tetramethyl-2，5-dihydro-1H-pyrrol-1-yloxy与半胱氨酸反应，提供汞连接的氮氧化物，在大多数螺旋表面与蛋白质的相互作用减少。因此，这些新的侧链可能是使用定点定向自旋标记进行结构确定的首选。
Previous studies have shown that the mobility of nitroxide side chains in a protein, inferred from the electron paramagnetic resonance (EPR) spectra, can be used to classify particular sites as helix surface sites, tertiary contact sites, buried sites, or loop sites. In addition, the sequence dependence of mobility can identify regular secondary structure. However, in the most widely used side chain, an apparent interaction of the nitroxide ring with the protein at some helix surface sites gives rise to EPR spectra degenerate with those at tertiary contact sites. In the present study, we use selected sites in T4 lysozyme to evaluate novel nitroxide side chains designed to resolve this degeneracy. The results indicate that the reagent 3-(methanesulfonylthiomethyl)-2,2,5,5-tetramethylpyrrolidin-1-yloxy reacts with cysteine to give a nitroxide side chain that has a high contrast in mobility between helix surface and tertiary contact sites, effectively resolving the degeneracy. The reagent 3-(iodomercuriomethyl)-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-1-yloxy reacts with cysteine to provide a mercury-linked nitroxide that also shows reduced interaction with the protein at most helix surface sites. Thus, these new side chains may be the preferred choices for structure determination using site-directed spin labeling.