Kumar Sujeet, Ruiz Natividad
Department of Microbiology, The Ohio State University, Columbus, OH, 43210, USA.
Bio Protoc. 2019 Jun 20;9(12):e3271. doi: 10.21769/BioProtoc.3271.
Transporters are dynamic membrane proteins that are essential to the physiology of cells. To function, transporters must cycle between various conformational states, so to understand their mechanistic details, it is critical to characterize how their structure changes during the transport cycle. One approach to studying the dynamics of transporters takes advantage of the chemistry of cysteine by using sulfhydryl-reactive, bi-functional cross-linkers to probe changes in the distance between two specific residues that have been substituted to cysteine. This approach is mostly used to study transporters , not in their natural cellular environment. Here we describe a protocol based on structure-guided cysteine cross-linking and proteolysis-coupled gel analysis to probe conformational changes of a target transporter in live cells. Although cross-linking approaches have been used to probe the proximity between transmembrane segments in membrane proteins , to our knowledge this protocol is the first to be used to interrogate transporter dynamics in cells. The use of this protocol is optimal for proteins with known or modeled structures to guide the replacement of specific residues with cysteines and the selection of cross-linking agents with various spacer arm lengths. This protocol allows for discriminating easily cross-linked and uncross-linked species and does not require the often difficult or unavailable reconstitution of transport activity in an system. In addition, this protocol could be used to probe the conformation of transporters in cells treated with transport inhibitors in order to better understand their mechanism of action, and potentially dynamic interactions between domains in proteins that are not transporters.
转运蛋白是对细胞生理功能至关重要的动态膜蛋白。为了发挥功能，转运蛋白必须在各种构象状态之间循环，因此，要了解其机制细节，关键在于表征其在转运循环过程中的结构变化。一种研究转运蛋白动力学的方法利用了半胱氨酸的化学性质，通过使用巯基反应性双功能交联剂来探测已被替换为半胱氨酸的两个特定残基之间距离的变化。这种方法主要用于研究转运蛋白，而非在其天然细胞环境中。在此，我们描述了一种基于结构导向的半胱氨酸交联和蛋白水解偶联凝胶分析的方案，用于探测活细胞中目标转运蛋白的构象变化。尽管交联方法已被用于探测膜蛋白中跨膜片段之间的接近程度，但据我们所知，该方案是首个用于探究细胞中转运蛋白动力学的方法。对于具有已知或建模结构的蛋白质，使用该方案可优化特定残基被半胱氨酸替换以及选择具有不同间隔臂长度的交联剂。该方案能够轻松区分易交联和未交联的物种，并且不需要在体外系统中经常难以实现或无法实现的转运活性重建。此外，该方案可用于探测经转运抑制剂处理的细胞中转运蛋白的构象，以便更好地理解其作用机制，以及潜在地了解非转运蛋白的蛋白质结构域之间的动态相互作用。
