title: Single-molecule spectroscopy of protein conformational dynamics in live eukaryotic cells.
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.
authors.affiliation: Department of Biochemistry, University of Zurich, Zurich, Switzerland.
authors.forename: Iwo
authors.initials: I
authors.lastname: König
authors.affiliation: Department of Biochemistry, University of Zurich, Zurich, Switzerland.
authors.forename: Arash
authors.initials: A
authors.lastname: Zarrine-Afsar
authors.affiliation: Department of Biochemistry, University of Zurich, Zurich, Switzerland.
authors.forename: Mikayel
authors.initials: M
authors.lastname: Aznauryan
authors.affiliation: Department of Biochemistry, University of Zurich, Zurich, Switzerland.
authors.forename: Andrea
authors.initials: A
authors.lastname: Soranno
authors.affiliation: Department of Biochemistry, University of Zurich, Zurich, Switzerland.
authors.forename: Bengt
authors.initials: B
authors.lastname: Wunderlich
authors.affiliation: Department of Biochemistry, University of Zurich, Zurich, Switzerland.
authors.forename: Fabian
authors.initials: F
authors.lastname: Dingfelder
authors.affiliation: Department of Biochemistry, University of Zurich, Zurich, Switzerland.
authors.forename: Jakob C
authors.initials: JC
authors.lastname: Stüber
authors.affiliation: Department of Biochemistry, University of Zurich, Zurich, Switzerland.
authors.forename: Andreas
authors.initials: A
authors.lastname: Plückthun
authors.affiliation: Department of Biochemistry, University of Zurich, Zurich, Switzerland.
authors.forename: Daniel
authors.initials: D
authors.lastname: Nettels
authors.affiliation: Department of Biochemistry, University of Zurich, Zurich, Switzerland.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Schuler
mesh_terms: D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism; D016000:Cluster Analysis; D003432:Cross-Linking Reagents / Q000737:chemistry; D004252:DNA Mutational Analysis; D023281:Genomics; D006151:Guanosine / Q000031:analogs & derivatives / Q000737:chemistry; D057809:HEK293 Cells; D006801:Humans; D008099:Liver / Q000378:metabolism; D051379:Mice; D016296:Mutagenesis; D009154:Mutation; D011487:Protein Conformation; D012333:RNA, Messenger / Q000378:metabolism; D020537:RNA, Small Nucleolar / Q000378:metabolism; D048348:Reverse Transcription; D013053:Spectrophotometry / Q000379:methods*; D014466:Ultraviolet Rays
chemical_list: D003432:Cross-Linking Reagents; D012333:RNA, Messenger; D020537:RNA, Small Nucleolar; D006151:Guanosine; C016578:7-methylguanosine