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Labeling of Proteins for Single-Molecule Fluorescence Spectroscopy

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Protein Folding

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2376))

Abstract

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.

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Acknowledgments

We thank Dr. Serge Chesnov and the Functional Genomics Center Zurich for expert mass spectrometry and MS data analysis, Fabian Dingfelder for contributing chromatograms and single-molecule data on ClyA, Erik D. Holmstrom for providing single-molecule data on NCBD, and Daniel Nettels for providing data analysis software and for helpful discussions.

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Author notes

  1. Franziska Zosel

    Present address: Novo Nordisk A/S, Måløv, Denmark

Authors and Affiliations

  1. Department of Biochemistry, University of Zurich, Zurich, Switzerland

    Franziska Zosel, Andrea Holla & Benjamin Schuler

  2. Department of Physics, University of Zurich, Zurich, Switzerland

    Benjamin Schuler

Authors
  1. Franziska Zosel
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  2. Andrea Holla
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  3. Benjamin Schuler
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Corresponding author

Correspondence to Benjamin Schuler .

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Editors and Affiliations

  1. Department of Bioengineering and Center for Cellular and Biomolecular Machines, University of California, Merced, Merced, CA, USA

    Victor Muñoz

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Zosel, F., Holla, A., Schuler, B. (2022). Labeling of Proteins for Single-Molecule Fluorescence Spectroscopy. In: Muñoz, V. (eds) Protein Folding. Methods in Molecular Biology, vol 2376. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1716-8_12

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  • DOI: https://doi.org/10.1007/978-1-0716-1716-8_12

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1715-1

  • Online ISBN: 978-1-0716-1716-8

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