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首页> 外文期刊>Methods: A Companion to Methods in Enzymology >Analysis of Ras:RasGEF interactions by phage display and static multi-angle light scattering.
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Analysis of Ras:RasGEF interactions by phage display and static multi-angle light scattering.

机译：通过噬菌体展示和静态多角度光散射分析Ras：RasGEF相互作用。

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摘要

Molecular switches such as small GTPases of the Ras family cycle between inactive GDP-bound and active GTP-bound states. Their essential role in controlling development and cell homeostasis requires mechanisms which determine amplitude and timing of activation. This is achieved in part by the action of guanine nucleotide exchange factors, which function as highly controlled enzymes whose activity relies on spatial segregation and intra- and intermolecular regulation. Here, we describe two experimental methodologies that permit the identification and characterization of GTPase binding sites on activators by assaying complex formation within a broad range of affinities. In the first assay system, proteins presented on the surface of filamentous phage are used to probe affinity determinants of protein-protein interactions. In this application, a protein-displayed phage library is generated by random mutagenesis and a plate-based selection is performed to identify mutations that confer higher binding affinity to an immobilized target. The second method uses light scattering as a tool for measuring the molecular weight, stoichiometry, and polydispersity of protein complexes in solution. In this application, conventional gel filtration chromatography provides initial fractionation, and in-line light scattering measurements allow accurate determination of molar masses of the eluent. This technique also provides information about conformational homogeneity which can be used as a quality
机译：分子开关，例如Ras家族的小GTPases，在无效的GDP约束状态和有效的GTP约束状态之间循环。它们在控制发育和细胞稳态中的重要作用需要确定激活幅度和时机的机制。这部分地通过鸟嘌呤核苷酸交换因子的作用来实现，所述鸟嘌呤核苷酸交换因子起高度受控的酶的作用，其活性依赖于空间分离以及分子内和分子间调节。在这里，我们描述了两种实验方法，可以通过在广泛的亲和力范围内检测复杂的形成物来鉴定和表征活化剂上的GTPase结合位点。在第一个测定系统中，存在于丝状噬菌体表面的蛋白质被用来探测蛋白质-蛋白质相互作用的亲和力决定因素。在本申请中，通过随机诱变生成蛋白质展示的噬菌体文库，并进行基于板的选择以鉴定赋予固定目标更高结合亲和力的突变。第二种方法使用光散射作为测量溶液中蛋白质复合物的分子量，化学计量和多分散性的工具。在此应用中，常规的凝胶过滤色谱可提供初始分级分离，并且在线光散射测量可准确确定洗脱液的摩尔质量。此技术还提供有关构象同质性的信息，可以用作质量信息

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