Accurate Transfer Efficiencies, Distance Distributions, and Ensembles of Unfolded and Intrinsically Disordered Proteins From Single-Molecule FRET.

Holmstrom ED; Holla A; Zheng W; Nettels D; Best RB; Schuler B

doi:10.1016/bs.mie.2018.09.030

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Journal article

Accurate Transfer Efficiencies, Distance Distributions, and Ensembles of Unfolded and Intrinsically Disordered Proteins From Single-Molecule FRET.

Holmstrom ED Department of Biochemistry, University of Zurich, Zurich, Switzerland. Electronic address: e.holmstrom@bioc.uzh.ch.
Holla A Department of Biochemistry, University of Zurich, Zurich, Switzerland.
Zheng W College of Integrative Sciences and Arts, Arizona State University, Mesa, AZ, United States. Electronic address: wenweizheng@asu.edu.
Nettels D Department of Biochemistry, University of Zurich, Zurich, Switzerland.
Best RB Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, United States. Electronic address: robertbe@helix.nih.gov.
Schuler B Department of Biochemistry, University of Zurich, Zurich, Switzerland; Department of Physics, University of Zurich, Zurich, Switzerland. Electronic address: schuler@bioc.uzh.ch.

2018-11-26

Published in:

Methods in enzymology. - 2018

Förster resonance energy transfer

Intrinsically disordered proteins

Fluorescence Resonance Energy Transfer

Intrinsically Disordered Proteins

English Intrinsically disordered proteins (IDPs) sample structurally diverse ensembles. Characterizing the underlying distributions of conformations is a key step toward understanding the structural and functional properties of IDPs. One increasingly popular method for obtaining quantitative information on intramolecular distances and distributions is single-molecule Förster resonance energy transfer (FRET). Here we describe two essential elements of the quantitative analysis of single-molecule FRET data of IDPs: the sample-specific calibration of the single-molecule instrument that is required for determining accurate transfer efficiencies, and the use of state-of-the-art methods for inferring accurate distance distributions from these transfer efficiencies. First, we illustrate how to quantify the correction factors for instrument calibration with alternating donor and acceptor excitation measurements of labeled samples spanning a wide range of transfer efficiencies. Second, we show how to infer distance distributions based on suitably parameterized simple polymer models, and how to obtain conformational ensembles from Bayesian reweighting of molecular simulations or from parameter optimization in simplified coarse-grained models.

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