Department of Biochemistry, University of Zurich, Zurich, Switzerland.
Novo Nordisk A/S, Måløv, Denmark.
Methods Mol Biol. 2022;2376:207-233. doi: 10.1007/978-1-0716-1716-8_12.
Single-molecule fluorescence spectroscopy has become an important technique for studying the conformational dynamics and folding of proteins. A key step for performing such experiments is the availability of high-quality samples. This chapter describes a simple and widely applicable strategy for preparing proteins that are site-specifically labeled with a donor and an acceptor dye for single-molecule Förster resonance energy transfer (FRET) experiments. The method is based on introducing two cysteine residues that are labeled with maleimide-functionalized fluorophores, combined with high-resolution chromatography. We discuss how to optimize site-specific labeling even in the absence of orthogonal coupling chemistry and present purification strategies that are suitable for samples ranging from intrinsically disordered proteins to large folded proteins. We also discuss common problems in protein labeling, how to avoid them, and how to stringently control sample quality.
单分子荧光光谱学已成为研究蛋白质构象动力学和折叠的重要技术。进行此类实验的关键步骤是获得高质量的样品。本章描述了一种简单且广泛适用的策略，用于制备在特定位置标记有供体和受体染料的蛋白质，以进行单分子Förster 共振能量转移（FRET）实验。该方法基于引入两个半胱氨酸残基，这些残基用马来酰亚胺功能化的荧光团标记，结合高分辨率色谱。我们讨论了如何优化甚至在没有正交偶联化学的情况下进行特异性标记，并提出了适用于从无规卷曲蛋白到大型折叠蛋白等各种样品的纯化策略。我们还讨论了蛋白质标记中的常见问题，如何避免这些问题以及如何严格控制样品质量。
