几个关键的生物事件在结合伙伴之间的瞬时相互作用中采用“命中即跑”策略。在某些情况下,其中一个结合伴侣的无序性质严重阻碍了共结晶的成功,往往导致只有一个合作伙伴的结晶。这里,我们讨论了一种捕获弱和瞬时蛋白质相互作用以进行结晶的方法。这种方法需要至少一个相互作用配偶体的结构细节和结合知识,以使用最佳大小的接头将蛋白质的已知最小结合区(肽)对接到另一个上。在结晶之前,应验证纯化的连接构建体的完整折叠和稳定性。在结构确定之后,结构指导的功能研究是独立的,全长未连接的蛋白质,以验证连接复合物的发现。我们设计了这种方法,然后使用内在无序的24个氨基酸的最小结合区域验证了其功效,神经元特异性底物,神经颗粒素和神经调节素,通过Gly链接器加入到他们的互动伙伴,钙调素.此外,报道的独立全长蛋白质的功能研究证实了连接肽复合物的发现。根据我们的研究,结合配套文献,我们建议优化的接头可以提供一个环境来模拟结合伴侣之间的自然相互作用,并为结构研究提供了有用的策略,以捕获与几种生物过程有关的弱和瞬态相互作用。
Several key biological events adopt a \"hit-and-run\" strategy in their transient interactions between binding partners. In some instances, the disordered nature of one of the binding partners severely hampers the success of co-crystallization, often leading to the crystallization of just one of the partners. Here, we discuss a method to trap weak and transient protein interactions for crystallization. This approach requires the structural details of at least one of the interacting partners and binding knowledge to dock the known minimum binding region (peptide) of the protein onto the other using an optimal-sized linker. Prior to crystallization, the purified linked construct should be verified for its intact folding and stability. Following structure determination, structure-guided functional studies are performed with independent, full-length unlinked proteins to validate the findings of the linked complex. We designed this approach and then validated its efficacy using a 24 amino acid minimum binding region of the intrinsically disordered, neuron-specific substrates, Neurogranin and Neuromodulin, joined via a Gly-linker to their interacting partner, Calmodulin. Moreover, the reported functional studies with independent full-length proteins confirmed the findings of the linked peptide complexes. Based on our studies, and in combination with the supporting literature, we suggest that optimized linkers can provide an environment to mimic the natural interactions between binding partners, and offer a useful strategy for structural studies to trap weak and transient interactions involved in several biological processes.