Labeling of Proteins for Single-Molecule Fluorescence Spectroscopy.

Autor:	Zosel F; Department of Biochemistry, University of Zurich, Zurich, Switzerland.; Novo Nordisk A/S, Måløv, Denmark., Holla A; Department of Biochemistry, University of Zurich, Zurich, Switzerland., Schuler B; Department of Biochemistry, University of Zurich, Zurich, Switzerland. schuler@bioc.uzh.ch.; Department of Physics, University of Zurich, Zurich, Switzerland. schuler@bioc.uzh.ch.
Jazyk:	angličtina
Zdroj:	Methods in molecular biology (Clifton, N.J.) [Methods Mol Biol] 2022; Vol. 2376, pp. 207-233.
DOI:	10.1007/978-1-0716-1716-8_12
Abstrakt:	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. (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)
Databáze:	MEDLINE
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