Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, California.
Department of Biology and Biological Engineering, Division of Chemical Biology, Chalmers Institute of Technology, Gothenburg, Sweden.
Curr Protoc Chem Biol. 2020 Mar;12(1):e80. doi: 10.1002/cpch.80.
Over the past few decades, numerous examples have demonstrated that intrinsic disorder in proteins lies at the heart of many vital processes, including transcriptional regulation, stress response, cellular signaling, and most recently protein liquid-liquid phase separation. The so-called intrinsically disordered proteins (IDPs) involved in these processes have presented a challenge to the classic protein "structure-function paradigm," as their functions do not necessarily involve well-defined structures. Understanding the mechanisms of IDP function is likewise challenging because traditional structure determination methods often fail with such proteins or provide little information about the diverse array of structures that can be related to different functions of a single IDP. Single-molecule fluorescence methods can overcome this ensemble-average masking, allowing the resolution of subpopulations and dynamics and thus providing invaluable insights into IDPs and their function. In this protocol, we describe a ratiometric single-molecule Förster resonance energy transfer (smFRET) routine that permits the investigation of IDP conformational subpopulations and dynamics. We note that this is a basic protocol, and we provide brief information and references for more complex analysis schemes available for in-depth characterization. This protocol covers optical setup preparation and protein handling and provides insights into experimental design and outcomes, together with background information about theory and a brief discussion of troubleshooting. © 2020 by John Wiley & Sons, Inc. Basic Protocol: Ratiometric smFRET detection and analysis of IDPs Support Protocol 1: Fluorophore labeling of a protein through maleimide chemistry Support Protocol 2: Sample chamber preparation Support Protocol 3: Determination of direct excitation of acceptor by donor excitation and leakage of donor emission to acceptor emission channel.
在过去的几十年中，许多例子表明蛋白质中的内源性无序处于许多重要过程的核心，包括转录调控、应激反应、细胞信号转导，以及最近的蛋白质液-液相分离。涉及这些过程的所谓的固有无序蛋白质 (IDP) 对经典的蛋白质“结构-功能范式”提出了挑战，因为它们的功能不一定涉及明确的结构。理解 IDP 功能的机制同样具有挑战性，因为传统的结构确定方法通常无法用于此类蛋白质，或者提供的有关可以与单个 IDP 的不同功能相关的各种结构的信息很少。单分子荧光方法可以克服这种整体平均掩蔽，从而可以解析亚群体和动力学，从而为 IDP 及其功能提供宝贵的见解。在本协议中，我们描述了一种比率单分子Förster 共振能量转移 (smFRET) 常规方法，该方法允许研究 IDP 构象亚群体和动力学。我们注意到，这是一个基本协议，并且我们为更复杂的分析方案提供了简要信息和参考，这些方案可用于深入表征。该协议涵盖了光学设置准备和蛋白质处理，并提供了有关实验设计和结果的见解，以及有关理论的背景信息和对故障排除的简要讨论。 2020 年，John Wiley & Sons，Inc. 基本方案：通过马来酰亚胺化学对 IDP 进行比率 smFRET 检测和分析 支持方案 1：通过马来酰亚胺化学对蛋白质进行荧光标记 支持方案 2：样品池制备 支持方案 3：通过供体激发确定对受体的直接激发以及供体发射漏到受体发射通道。
