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Single-Molecule Fluorescence Spectroscopy of Intrinsically Disordered Proteins

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Fluorescence Spectroscopy and Microscopy in Biology

Part of the book series: Springer Series on Fluorescence ((SS FLUOR,volume 20))

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Abstract

The past two decades have seen a substantial leap forward in our understanding of intrinsically disordered proteins, in terms of both thermodynamics and dynamics, but also in terms of structural ensembles. From understanding the principles and biological importance of their solvent pliability up to characterizing their dynamics including an identification of the molecular origins of internal friction, single-molecule FRET experiments have been an important driver of this progress. By now, the methods and analysis tools in single-molecule FRET have grown to an extensive toolbox that allows a straightforward comparison of experiments with analytical theories and results of molecular simulations. This chapter summarizes the technologies behind single-molecule FRET experiments and molecular simulations together with the key findings on intrinsically disordered proteins.

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Notes

  1. 1.

    Here, δ indicates the Dirac delta function.

  2. 2.

    De Gennes derived his model for the radius of gyration (rG) of the polymer. Since the distribution of radii of gyration for a Gaussian chain is not known in closed analytic form, he used the approximation P(α) ∝ α3 exp (−3α2/2) where α = rG/RG, ideal. Notably, compared to donor–acceptor distances measured with smFRET, the radius of gyration is by far the better quantity to construct a mean-field theory due to its direct link to the monomer-density of a chain. The long-known fact has more recently gained renewed attention in the so-called FRET-SAXS controversy [see Refs. 22 and 77].

  3. 3.

    Here, photons from each color are randomly distributed to two detectors.

  4. 4.

    For example, after FRET from donor to acceptor, the acceptor is in the excited state. If the donor is re-exited before the acceptor relaxes to the ground state, both dyes will be in the excited state.

  5. 5.

    Importantly, the intrinsic diffusion of donor and acceptor relative to each other should not be confused with the translational diffusion of the whole molecule.

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Acknowledgments

H.H. thanks the Israel Science Foundation (ISF 2253/18) and the European Research Council (ERC-CoG 864578). W.Z. acknowledges the support from the National Science Foundation (MCB-2015030) and the National Institutes of Health (R35GM146814).

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Authors and Affiliations

  1. Department of Chemical and Structural Biology, Weizmann Institute of Science, Rehovot, Israel

    Hagen Hofmann

  2. College of Integrative Sciences and Arts, Arizona State University, Mesa, AZ, USA

    Wenwei Zheng

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  1. Hagen Hofmann
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  2. Wenwei Zheng
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Correspondence to Hagen Hofmann .

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  1. J. Heyrovský Institute of Physical Chemistry, Prague, Czech Republic

    Radek Šachl

  2. J. Heyrovský Institute of Physical Chemistry, Prague, Czech Republic

    Mariana Amaro

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Hofmann, H., Zheng, W. (2022). Single-Molecule Fluorescence Spectroscopy of Intrinsically Disordered Proteins. In: Šachl, R., Amaro, M. (eds) Fluorescence Spectroscopy and Microscopy in Biology. Springer Series on Fluorescence, vol 20. Springer, Cham. https://doi.org/10.1007/4243_2022_38

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