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首页> 外文期刊>Biochemistry >Specific cross-linking of lys233 and cys235 in the mu opioid receptor by a reporter affinity label
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Specific cross-linking of lys233 and cys235 in the mu opioid receptor by a reporter affinity label

机译：通过报告亲和标记，μ阿片受体中lys233和cys235的特定交联

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摘要

The first example of the use of a reporter affinity label (NNA) that contains a fluorogenic naphthalene dialdehyde moiety to identify neighboring lysine and cysteine residues at a recognition site is described. The opioid receptors have served as the proof-of-concept because they contain multiple lysine and cysteine residues. The kinetics of isoindole formation resulting from covalent binding of NNA to wild-type and mutant opioid receptors were followed in cultured cells using flow cytometry. The finding that NNA bound to mutant mu opioid receptors (K233R and C235S) without producing specific fluorescence enhancement suggested that covalent bonding occurred at these positions to produce an isoindole fluorophore in the wild-type mu receptor. The similar kinetics of fluorophore formation for wild-type mu, delta, and kappa opioid receptors suggest that these conserved residues are the cross-linking sites in all three types of opioid receptors. The combined utilization of a reporter affinity label and site-directed mutagenesis offers a more expeditious method of identifying cross-linking at a recognition site when compared to classical procedures.
机译：描述了使用报道分子亲和标记物（NNA）的第一个例子，该报道物亲和标记物包含荧光萘二醛部分以识别识别位点附近的赖氨酸和半胱氨酸残基。阿片受体已成为概念证明，因为它们含有多个赖氨酸和半胱氨酸残基。使用流式细胞仪追踪培养细胞中NNA与野生型和突变型阿片受体共价结合产生的异吲哚形成的动力学。 NNA与突变型mu阿片受体（K233R和C235S）结合而不产生特异性荧光增强的发现表明，在这些位置上发生了共价键合，从而在野生型mu受体中产生了异吲哚荧光团。野生型mu，delta和kappa类阿片受体的荧光团形成动力学相似，表明这些保守残基是所有三种类型阿片受体的交联位点。与传统方法相比，结合使用报告子亲和力标签和定点诱变提供了一种更快速的方法来识别识别位点的交联。

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