A chemical proteomic probe for detecting dehydrogenases: catechol rhodanine.
A chemical proteomic probe for detecting dehydrogenases: catechol rhodanine.
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用于检测脱氢酶的化学蛋白质组探针：儿茶酚绕丹宁。
DOI：
10.1007/978-1-61779-364-6_5
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发表时间：
2012
期刊：
影响因子：
--
通讯作者：
Sem,DanielS
中科院分区：
文献类型：
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作者：
Ge,Xia;Sem,DanielS
蛋白质组固有的复杂性通常要求将其作为可管理的子集进行研究，称为亚蛋白质组。亚蛋白质组可以用多种方式定义，尽管实用的方法是基于活性部位的共同特征来定义它，这些特征导致共同的小分子配体(例如，辅因子或交叉反应药物先导)的结合。如此定义的亚蛋白质组可通过亲和层析，使用与树脂连接的公共配基来纯化。亚蛋白质组的亲和纯化将在下一章中描述。然后，可以使用公共配基探针来分析该亚蛋白质组，例如可以用来在天然凝胶中对亚蛋白质组成员进行染色的荧光公共配基。在这里，我们描述了这样一种荧光探针，基于与脱氢酶结合的儿茶酚罗丹宁乙酸(CRAA)配体。CRAa配体是荧光的，在pH>；7时与脱氢酶结合，因此可以有效地用于对天然凝胶中的脱氢酶进行染色，以确定混合物中的哪一种蛋白质是脱氢酶。此外，如果设计抑制剂来靶向这些脱氢酶中的一个或多个，则可以竞争分析的形式进行CRAA染色，无论是否有抑制剂，以评估靶向脱氢酶的抑制剂的选择性。最后，CraA探针是脱氢酶的特权支架，因此可以很容易地进行修饰，以增加对给定脱氢酶的亲和力。
Inherent complexity of the proteome often demands that it be studied as manageable subsets, termed subproteomes. A subproteome can be defined in a number of ways, although a pragmatic approach is to define it based on common features in an active site that lead to binding of a common small molecule ligand (e.g., a cofactor or a cross-reactive drug lead). The subproteome, so defined, can be purified using that common ligand tethered to a resin, with affinity chromatography. Affinity purification of a subproteome is described in the next chapter. That subproteome can then be analyzed using a common ligand probe, such as a fluorescent common ligand that can be used to stain members of the subproteome in a native gel. Here, we describe such a fluorescent probe, based on a catechol rhodanine acetic acid (CRAA) ligand that binds to dehydrogenases. The CRAA ligand is fluorescent and binds to dehydrogenases at pH > 7, and hence can be used effectively to stain dehydrogenases in native gels to identify what subset of proteins in a mixture are dehydrogenases. Furthermore, if one is designing inhibitors to target one or more of these dehydrogenases, the CRAA staining can be performed in a competitive assay format, with or without inhibitor, to assess the selectivity of the inhibitor for the targeted dehydrogenase. Finally, the CRAA probe is a privileged scaffold for dehydrogenases, and hence can easily be modified to increase affinity for a given dehydrogenase.