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

Iwo König, Arash Zarrine-Afsar, Mikayel Aznauryan, Andrea Soranno, Bengt Wunderlich, Fabian Dingfelder, Jakob C. Stüber, Andreas Plückthun, Daniel Nettels, Benjamin Schuler

Research output: Contribution to journalArticlepeer-review

179 Scopus citations


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.

Original languageEnglish
Pages (from-to)773-779
Number of pages7
JournalNature Methods
Issue number8
StatePublished - Jul 30 2015


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