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Intrinsically Disordered Protein Analysis pp 21–45Cite as

Application of Confocal Single-Molecule FRET to Intrinsically Disordered Proteins

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 896))

Abstract

Intrinsically disordered proteins (IDPs) are characterized by a large degree of conformational heterogeneity. In such cases, classical experimental methods often yield only mean values, averaged over the entire ensemble of molecules. The microscopic distributions of conformations, trajectories, or sequences of events often remain unknown, and with them the underlying molecular mechanisms. Signal averaging can be avoided by observing individual molecules. A particularly versatile method is highly sensitive fluorescence detection. In combination with Förster resonance energy transfer (FRET), distances and conformational dynamics can be investigated in single molecules. This chapter introduces the practical aspects of applying confocal single-molecule FRET experiments to the study of IDPs.

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Notes

  1. 1.

    The limiting factor for the time resolution currently is the jitter of APDs in the range of 100 ps.

  2. 2.

    The mean squared distance between the attachment points can be estimated from \( \left\langle {{r^2}} \right\rangle = 2{l_{\rm{p}}}{l_{\rm{c}}} \), where l p is the persistence length of the IDP, typically in the range between 0.2 and 0.4 nm (19, 39) in the absence of strong charge repulsion (40), and l c is the contour length of the segment, which can be calculated as N∙0.38 nm, where N corresponds to the number of peptide bonds in the segment (19). The observed transfer efficiency can then be estimated from Eq. (5).

  3. 3.

    For preliminary screens or in case the sequential labeling procedure is not feasible, both dyes can be added simultaneously. In many cases, the fraction of molecules that contain only donor chromophores can be reduced by empirically varying the ratio of donor and acceptor dye in the reaction. Even relatively large proportions of “donor only” molecules can be tolerated in single molecule experiments because of the separation of subpopulations, but a high-quality sample preparation will simplify data acquisition, analysis, and interpretation greatly.

  4. 4.

    \( \int_a^{{{l_{\rm{c}}}}} {P(r)} {\hbox{d}}r = 1 \).

  5. 5.

    Note that the averaging has to be done over the transfer rate constant k t, i.e. \( \left\langle E\, \right\rangle = {{1} / {{( {1 + {k_{\rm{D}}}/\int_a^{{{l_{\rm{c}}}}} {{k_{\rm{t}}}(r)P(r){\hbox{d}}r} })}}} \), where \( {k_{\rm{t}}}(r) = {k_{\rm{D}}}{\left( {{{{{R_0}}} \left/ {r} \right.}} \right)^6} \), and k D is the fluorescence decay rate constant of the donor in the absence of the acceptor.

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Acknowledgments

This work has been supported by the Swiss National Science Foundation, the Swiss National Center of Competence in Research for Structural Biology, and a Starting Researcher Grant by the European Research Council.

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Authors and Affiliations

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

    Benjamin Schuler, Sonja Müller-Späth, Andrea Soranno & Daniel Nettels

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  1. Benjamin Schuler
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Correspondence to Benjamin Schuler .

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  1. DEPT OF MOLECULAR MED, College of Medicine, University of South Florida, BRUCE B DOWNS BLVD 12901, TAMPA, 33612, Florida, USA

    Vladimir N. Uversky

  2. Dept. Biochemistry & Molecular Biology, Center for Computational Biology &, Indiana University, West 10th Street 410, Indianapolis, 46202, Indiana, USA

    A. Keith Dunker

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Schuler, B., Müller-Späth, S., Soranno, A., Nettels, D. (2012). Application of Confocal Single-Molecule FRET to Intrinsically Disordered Proteins. In: Uversky, V., Dunker, A. (eds) Intrinsically Disordered Protein Analysis. Methods in Molecular Biology, vol 896. Springer, New York, NY. https://doi.org/10.1007/978-1-4614-3704-8_2

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