Mapping of Protein Interfaces in Live Cells Using Genetically Encoded Crosslinkers.
Mapping of Protein Interfaces in Live Cells Using Genetically Encoded Crosslinkers.
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DOI：
10.1007/978-1-4939-7574-7_14
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发表时间：
2018
影响因子：
--
通讯作者：
L. Seidel;Irene Coin
中科院分区：
文献类型：
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作者：
L. Seidel;Irene Coin
了解蛋白质-蛋白质相互作用的拓扑结构在生物医学领域是一个至关重要的问题。生物物理方法，如x射线晶体学和核磁共振，只能详细研究在人工环境中重建的分离蛋白复合物。需要替代的方法来研究蛋白质在生理背景下的相互作用，以及表征逃避直接结构表征的蛋白质复合物。我们在这里描述了一种一般策略，直接在活体哺乳动物细胞中研究分子水平上的蛋白质相互作用，这是基于光交联和化学交联非规范氨基酸的遗传结合。首先，光交联氨基酸被用来绘制假定的相互作用表面，并确定蛋白质的哪个位置接近相关伙伴。在第二步中，属于结合界面的残基子集被化学交联剂取代，该交联剂选择性地与放置在相互作用伙伴中的近端半胱氨酸发生反应。这允许确定分子间空间约束，为建立精确的分子模型提供基础。在本章中，我们详细说明了这一实验策略的应用，以揭示40粒神经肽激素尿皮质素1与其B类g蛋白偶联受体（促肾上腺皮质激素释放因子受体1型）的结合方式。该方法原则上适用于与蛋白质类型和大小无关的任何蛋白质复合物，采用已建立的非规范氨基酸诱变技术，并且在任何分子生物学实验室都是可行的。
Understanding the topology of protein-protein interactions is a matter of fundamental importance in the biomedical field. Biophysical approaches such as X-ray crystallography and nuclear magnetic resonance can investigate in detail only isolated protein complexes that are reconstituted in an artificial environment. Alternative methods are needed to investigate protein interactions in a physiological context, as well as to characterize protein complexes that elude the direct structural characterization. We describe here a general strategy to investigate protein interactions at the molecular level directly in the live mammalian cell, which is based on the genetic incorporation of photo- and chemical crosslinking noncanonical amino acids. First a photo-crosslinking amino acid is used to map putative interaction surfaces and determine which positions of a protein come into proximity of an associated partner. In a second step, the subset of residues that belong to the binding interface are substituted with a chemical crosslinker that reacts selectively with proximal cysteines strategically placed in the interaction partner. This allows determining inter-molecular spatial constraints that provide the basis for building accurate molecular models. In this chapter, we illustrate the detailed application of this experimental strategy to unravel the binding modus of the 40-mer neuropeptide hormone Urocortin1 to its class B G-protein coupled receptor, the corticotropin releasing factor receptor type 1. The approach is in principle applicable to any protein complex independent of protein type and size, employs established techniques of noncanonical amino acid mutagenesis, and is feasible in any molecular biology laboratory.