Header

UZH-Logo

Maintenance Infos

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


Holmstrom, Erik D; Holla, Andrea; Zheng, Wenwei; Nettels, Daniel; Best, Robert B; Schuler, Benjamin (2018). Accurate Transfer Efficiencies, Distance Distributions, and Ensembles of Unfolded and Intrinsically Disordered Proteins From Single-Molecule FRET. Methods in Enzymology, 611:287-325.

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.

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.

Statistics

Citations

Altmetrics

Downloads

1 download since deposited on 15 Feb 2019
1 download since 12 months
Detailed statistics

Additional indexing

Item Type:Journal Article, refereed, further contribution
Communities & Collections:04 Faculty of Medicine > Department of Biochemistry
07 Faculty of Science > Department of Biochemistry
Dewey Decimal Classification:570 Life sciences; biology
610 Medicine & health
Language:English
Date:2018
Deposited On:15 Feb 2019 09:01
Last Modified:15 Feb 2019 09:02
Publisher:Elsevier
ISSN:0076-6879
OA Status:Closed
Publisher DOI:https://doi.org/10.1016/bs.mie.2018.09.030
PubMed ID:30471690

Download