Labeling of Proteins for Single-Molecule Fluorescence Spectroscopy

• Published in: Methods in molecular biology (Clifton, N.J.) Vol. 2376; p. 207
• Main Authors: Zosel, Franziska, Holla, Andrea, Schuler, Benjamin
• Format: Journal Article
• Language: English
• Published: United States 2022
• Subjects: Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Intrinsically Disordered Proteins; Single Molecule Imaging; Spectrometry, Fluorescence; Fluorescence spectroscopy; RP-HPLC; Fluorescent dye; Förster resonance energy transfer; Cysteine; Intrinsically disordered proteins; Maleimide; Protein folding; Ion-exchange chromatography; Single-molecule detection; FRET; Labeling
• ISSN: 1940-6029
• 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.