Confocal single-molecule FRET for protein conformational dynamics

Yan Wen Tan, Jeffrey A. Hanson, Jhih Wei Chu, Haw Yang

Research output: Chapter in Book/Report/Conference proceedingChapter

7 Scopus citations

Abstract

Single-molecule FÓ§rster-type resonance energy transfer (smFRET) is a unique technique capable of following conformational motions of individual protein molecules. The direct observation of individual proteins provides rich information that would be washed away in ensemble measurements, hence opening up new avenues for establishing the protein structure-function relationships through dynamics. Retrieving dynamics information of biomolecular motions via smFRET, though, requires careful experiment design and rigorous treatment of single-molecule statistics. Here, we describe the rudimentary steps for an smFRET experiment, including sample preparation for the microscope, building of critical parts for single-molecule FRET detection, and a robust methodology for photon-by-photon data analysis.

Original languageEnglish (US)
Title of host publicationProtein Dynamics
Subtitle of host publicationMethods and Protocols
PublisherHumana Press Inc.
Pages51-62
Number of pages12
ISBN (Print)9781627036573
DOIs
StatePublished - 2014

Publication series

NameMethods in Molecular Biology
Volume1084

All Science Journal Classification (ASJC) codes

  • Genetics
  • Molecular Biology

Keywords

  • Model free
  • Poisson statistics
  • Protein immobilization
  • Single photon counting

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    Tan, Y. W., Hanson, J. A., Chu, J. W., & Yang, H. (2014). Confocal single-molecule FRET for protein conformational dynamics. In Protein Dynamics: Methods and Protocols (pp. 51-62). (Methods in Molecular Biology; Vol. 1084). Humana Press Inc.. https://doi.org/10.1007/978-1-62703-658-0_3