Application of linker technique to trap transiently interacting protein complexes for structural studies.
Application of linker technique to trap transiently interacting protein complexes for structural studies.
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DOI：
10.14440/jbm.2016.81
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发表时间：
2016
期刊：
影响因子：
--
通讯作者：
Sivaraman J
中科院分区：
文献类型：
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作者：
Reddy Chichili VP;Kumar V;Sivaraman J
蛋白质-蛋白质相互作用是控制多种生物过程的关键事件。我们已经开发并采用了一种方法来捕获瞬时相互作用的蛋白质复合物的结构研究，使用富含甘氨酸的接头融合相互作用的合作伙伴，其中之一是非结构化的。初始步骤包括等温滴定量热法，以确定非结构化蛋白质与其稳定结合伴侣相互作用的最小结合区域。随后进行计算分析以确定相互作用的近似位点并设计适当的接头长度。随后，使用尺寸排阻色谱法和动态光散射实验产生并表征融合构建体。然后通过结晶解析嵌合蛋白的结构，并通过用丙氨酸取代全长未连接蛋白的关键相互作用残基在体外和体内验证。该协议提供了机会，研究关键的和目前无法实现的瞬时蛋白质相互作用参与各种生物过程。
Protein-protein interactions are key events controlling several biological processes. We have developed and employed a method to trap transiently interacting protein complexes for structural studies using glycine-rich linkers to fuse interacting partners, one of which is unstructured. Initial steps involve isothermal titration calorimetry to identify the minimum binding region of the unstructured protein in its interaction with its stable binding partner. This is followed by computational analysis to identify the approximate site of the interaction and to design an appropriate linker length. Subsequently, fused constructs are generated and characterized using size exclusion chromatography and dynamic light scattering experiments. The structure of the chimeric protein is then solved by crystallization, and validated both in vitro and in vivo by substituting key interacting residues of the full length, unlinked proteins with alanine. This protocol offers the opportunity to study crucial and currently unattainable transient protein interactions involved in various biological processes.