Ensemble and single-molecule detected time-resolved FRET methods in studies of protein conformations and dynamics

Tomer Orevi, Eitan Lerner, Gil Rahamim, Dan Amir, Elisha Haas

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

14 Scopus citations

Abstract

Most proteins are nanomachines that are selected to execute specific functions and therefore should have some degree of flexibility. The driving force that excites specific motions of domains and smaller chain elements is the thermal fluctuations of the solvent bath which are channeled to selected modes of motions by the structural constraints. Consequently characterization of the ensembles of conformers of proteins and their dynamics should be expressed in statistical terms, i.e., determination of probability distributions of the various conformers. This can be achieved by measurements of time-resolved dynamic non-radiative excitation energy transfer (trFRET) within ensembles of site specifically labeled protein molecules. Distributions of intramolecular segmental end-to-end distances and their fast fluctuations can be determined, and fast and slow conformational transitions within selected sections of the molecule can be monitored and analyzed. Both ensemble and single-molecule detection methods can be applied for data collection. In combination with synchronization methods, time-resolved FRET was also used for studies of fast conformational transitions, in particular the folding/unfolding transitions.

Original languageAmerican English
Title of host publicationFluorescence Spectroscopy and Microscopy
Subtitle of host publicationMethods and Protocols
PublisherHumana Press Inc.
Pages113-169
Number of pages57
ISBN (Print)9781627036481
DOIs
StatePublished - 2014
Externally publishedYes

Publication series

NameMethods in Molecular Biology
Volume1076
ISSN (Print)1064-3745

Keywords

  • Distance distributions
  • Fast fluctuations and conformational transitions
  • Intramolecular diffusion coefficient
  • Protein conformational dynamics
  • Single-molecule detection
  • Site-specific labeling
  • Time-resolved FRET

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