A Multicolor Single-Molecule FRET Approach to Study Protein Dynamics and Interactions Simultaneously

M. Götz, P. Wortmann, S. Schmid, T. Hugel*

*Corresponding author for this work

Research output: Chapter in Book/Report/Conference proceedingChapterAcademicpeer-review

11 Citations (Scopus)


Single-molecule Förster resonance energy transfer (smFRET) is a versatile tool for studying biomolecules in a quantitative manner. Multiple conformations within and interactions between biomolecules can be detected and their kinetics can be determined. Thus, smFRET has become an essential tool in enzymology. Ordinary two-color smFRET experiments can provide only limited insight into the function of biological systems, which commonly consist of more than two components. A complete understanding of complex multicomponent biological systems requires correlated information on conformational rearrangements on the one hand and transient interactions with binding partners on the other. Multicolor smFRET experiments enable the direct observation of such correlated dynamics and interactions. Here we demonstrate the power and limitations of multicolor smFRET experiments including the description of a multicolor smFRET setup and data analysis. A general analytical procedure for multicolor smFRET data is presented and applied to the multicomponent heat shock protein 90 system. This allows us to identify microscopic states in transient complexes. Conformational dynamics and nucleotide binding are simultaneously detected, which is impossible using two-color smFRET. Additionally, their correlation is quantified using 3D ensemble hidden Markov analysis, in and out of equilibrium. This method is perfectly suited for protein systems that are much more sophisticated than previously studied DNA-based systems. By extending the application to biologically relevant systems, multicolor smFRET comes of age and provides a unique mechanistic insight into protein machines.

Original languageEnglish
Title of host publicationSingle-Molecule Enzymology: Fluorescence-Based and High-Throughput Methods
EditorsMaria Spies, Yann R. Chemla
PublisherAcademic Press Inc.
Number of pages30
ISBN (Print)9780128092675
Publication statusPublished - 2016
Externally publishedYes

Publication series

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


  • ATPase
  • Correlated kinetics
  • HMM
  • Hsp90
  • Multicolor FRET
  • Protein dynamics
  • Single molecule
  • TIRF


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