Header

UZH-Logo

Maintenance Infos

Single-molecule spectroscopy of protein conformational dynamics in live eukaryotic cells


König, Iwo; Zarrine-Afsar, Arash; Aznauryan, Mikayel; Soranno, Andrea; Wunderlich, Bengt; Dingfelder, Fabian; Stüber, Jakob C; Plückthun, Andreas; Nettels, Daniel; Schuler, Benjamin (2015). Single-molecule spectroscopy of protein conformational dynamics in live eukaryotic cells. Nature Methods, 12(8):773-779.

Abstract

Single-molecule methods have become widely used for quantifying the conformational heterogeneity and structural dynamics of biomolecules in vitro. Their application in vivo, however, has remained challenging owing to shortcomings in the design and reproducible delivery of labeled molecules, the range of applicable analysis methods, and suboptimal cell culture conditions. By addressing these limitations in an integrated approach, we demonstrate the feasibility of probing protein dynamics from milliseconds down to the nanosecond regime in live eukaryotic cells with confocal single-molecule Förster resonance energy transfer (FRET) spectroscopy. We illustrate the versatility of the approach by determining the dimensions and submicrosecond chain dynamics of an intrinsically disordered protein; by detecting even subtle changes in the temperature dependence of protein stability, including in-cell cold denaturation; and by quantifying the folding dynamics of a small protein. The methodology opens possibilities for assessing the effect of the cellular environment on biomolecular conformation, dynamics and function.

Abstract

Single-molecule methods have become widely used for quantifying the conformational heterogeneity and structural dynamics of biomolecules in vitro. Their application in vivo, however, has remained challenging owing to shortcomings in the design and reproducible delivery of labeled molecules, the range of applicable analysis methods, and suboptimal cell culture conditions. By addressing these limitations in an integrated approach, we demonstrate the feasibility of probing protein dynamics from milliseconds down to the nanosecond regime in live eukaryotic cells with confocal single-molecule Förster resonance energy transfer (FRET) spectroscopy. We illustrate the versatility of the approach by determining the dimensions and submicrosecond chain dynamics of an intrinsically disordered protein; by detecting even subtle changes in the temperature dependence of protein stability, including in-cell cold denaturation; and by quantifying the folding dynamics of a small protein. The methodology opens possibilities for assessing the effect of the cellular environment on biomolecular conformation, dynamics and function.

Statistics

Citations

Dimensions.ai Metrics
65 citations in Web of Science®
62 citations in Scopus®
78 citations in Microsoft Academic
Google Scholar™

Altmetrics

Downloads

1 download since deposited on 24 Sep 2015
0 downloads since 12 months
Detailed statistics

Additional indexing

Item Type:Journal Article, refereed, original work
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:August 2015
Deposited On:24 Sep 2015 10:18
Last Modified:16 Mar 2018 15:16
Publisher:Nature Publishing Group
ISSN:1548-7091
OA Status:Closed
Publisher DOI:https://doi.org/10.1038/nmeth.3475
PubMed ID:26147918

Download

Download PDF  'Single-molecule spectroscopy of protein conformational dynamics in live eukaryotic cells'.
Preview
Content: Accepted Version
Filetype: PDF
Size: 664kB
View at publisher