双氮氧化物标记的铜催化叠氮化物-炔烃环加成反应与非经典氨基酸用于电子顺磁共振波谱学。
Double Nitroxide Labeling by Copper-Catalyzed Azide-Alkyne Cycloadditions with Noncanonical Amino Acids for Electron Paramagnetic Resonance Spectroscopy.
机构信息
Department of Chemistry and Konstanz Research School Chemical Biology , University of Konstanz , Konstanz , Germany.
Faculty of Chemistry and Chemical Biology , TU Dortmund University , Dortmund , Germany.
出版信息
ACS Chem Biol. 2019 May 17;14(5):839-844. doi: 10.1021/acschembio.8b01111. Epub 2019 May 1.
Electron paramagnetic resonance spectroscopy in combination with site-directed spin labeling (SDSL) is an important tool to obtain long-range distance restraints for protein structural research. We here study a variety of azide- and alkyne-bearing noncanonical amino acids (ncAA) in terms of protein single- and double-incorporation efficiency via nonsense suppression, metabolic stability, yields of nitroxide labeling via copper-catalyzed [3 + 2] azide-alkyne cycloadditions (CuAAC), and spectroscopic properties in continuous-wave and double electron-electron resonance measurements. We identify para-ethynyl-l-phenylalanine and para-propargyloxy-l-phenylalanine as suitable ncAA for CuAAC-based SDSL that will complement current SDSL approaches, particularly in cases in which essential cysteines of a target protein prevent the use of sulfhydryl-reactive spin labels.
电子顺磁共振波谱与定点自旋标记（SDSL）相结合是获得蛋白质结构研究中长程距离约束的重要工具。我们在这里通过无义抑制研究了各种带有叠氮化物和炔烃的非天然氨基酸（ncAA）在蛋白质单掺入和双掺入效率方面的情况，包括代谢稳定性、通过铜催化的[3 + 2]叠氮-炔环加成反应（CuAAC）生成氮氧自由基标记的产率，以及在连续波和双电子电子共振测量中的光谱特性。我们确定对乙炔基-l-苯丙氨酸和对丙炔氧基-l-苯丙氨酸为适合基于 CuAAC 的 SDSL 的 ncAA，这将补充当前的 SDSL 方法，特别是在目标蛋白质的必需半胱氨酸阻止使用巯基反应性自旋标记的情况下。
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