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

Erik D. Holmstrom, Andrea Holla, Wenwei Zheng, Daniel Nettels, Robert B. Best, Benjamin Schuler

Research output: Chapter in Book/Report/Conference proceedingChapter

8 Scopus citations

Abstract

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.

Original languageEnglish (US)
Title of host publicationMethods in Enzymology
EditorsElizabeth Rhoades
PublisherAcademic Press Inc.
Pages287-325
Number of pages39
ISBN (Print)9780128156490
DOIs
StatePublished - Jan 1 2018
Externally publishedYes

Publication series

NameMethods in Enzymology
Volume611
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988

Keywords

  • Förster resonance energy transfer
  • Intrinsically disordered proteins
  • Molecular simulations
  • Polymers
  • Single-molecule spectroscopy

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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    Holmstrom, E. D., Holla, A., Zheng, W., Nettels, D., Best, R. B., & Schuler, B. (2018). Accurate Transfer Efficiencies, Distance Distributions, and Ensembles of Unfolded and Intrinsically Disordered Proteins From Single-Molecule FRET. In E. Rhoades (Ed.), Methods in Enzymology (pp. 287-325). (Methods in Enzymology; Vol. 611). Academic Press Inc.. https://doi.org/10.1016/bs.mie.2018.09.030