Site-directed spin labeling of proteins by chemical modification of engineered cysteine residues with the molecule MTSSL (1-oxyl-2,2,5,5-tetramethylpyrroline-3-methyl methanethiosulfonate) has been an invaluable tool for conducting double electron electron resonance (DEER) spectroscopy experiments. However, this method is generally limited to recombinant proteins with a limited number of reactive Cys residues that when modified will not impair protein function. Here, we present a method that allows for spin labeling of protein-nucleotide-binding sites by adenosine diphosphate (ADP) modified with a nitroxide moiety on the β-phosphate (ADP-β-S-SL). The synthesis of this ADP analog is straightforward and isolation of pure product is readily achieved on a standard reverse-phase high-performance liquid chromatography (HPLC) system. Furthermore, analyses of isolated ADP-β-S-SL by LC–mass spectrometry confirm that the molecule is very stable under ambient conditions. The crystal structure of ADP-β-S-SL bound to the ATP pocket of the histidine kinase CheA reveals specific targeting of the probe, whose nitroxide moiety is mobile on the protein surface. Continuous wave and pulsed-ESR measurements demonstrate the capability of ADP-β-S-SL to report on active site environment and provide reliable DEER distance constraints.

中文翻译：

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用于 ATP 结合蛋白的 ESR 光谱的核苷酸自旋标记

通过用分子 MTSSL（1-oxyl-2,2,5,5-tetramethylpyrroline-3-methyl methanethiosulfonate）对工程半胱氨酸残基进行化学修饰，对蛋白质进行定点自旋标记已成为进行双电子共振的宝贵工具。 DEER) 光谱实验。然而，这种方法通常仅限于具有有限数量的反应性 Cys 残基的重组蛋白质，这些残基在修饰时不会损害蛋白质功能。在这里，我们提出了一种方法，该方法允许通过在 β-磷酸（ADP-β-S-SL）上用氮氧部分修饰的二磷酸腺苷（ADP）对蛋白质-核苷酸结合位点进行自旋标记。这种 ADP 类似物的合成很简单，在标准的反相高效液相色谱 (HPLC) 系统上很容易实现纯产品的分离。此外，通过 LC-质谱法对分离的 ADP-β-S-SL 的分析证实该分子在环境条件下非常稳定。ADP-β-S-SL 的晶体结构与组氨酸激酶 CheA 的 ATP 口袋结合，揭示了探针的特异性靶向，其氮氧化物部分可在蛋白质表面移动。连续波和脉冲 ESR 测量证明了 ADP-β-S-SL 能够报告活动场地环境并提供可靠的 DEER 距离约束。