Nonfluorescent quenchers to correlate single-molecule conformational and compositional dynamics

J Am Chem Soc. 2012 Apr 4;134(13):5734-7. doi: 10.1021/ja2119964. Epub 2012 Mar 23.

Abstract

Single-molecule Förster resonance energy transfer (smFRET) is a powerful method for studying the conformational dynamics of a biomolecule in real-time. However, studying how interacting ligands correlate with and regulate the conformational dynamics of the biomolecule is extremely challenging because of the availability of a limited number of fluorescent dyes with both high quantum yield and minimal spectral overlap. Here we report the use of a nonfluorescent quencher (Black Hole Quencher, BHQ) as an acceptor for smFRET. Using a Cy3/BHQ pair, we can accurately follow conformational changes of the ribosome during elongation in real time. We demonstrate the application of single-color FRET to correlate the conformational dynamics of the ribosome with the compositional dynamics of tRNA. We use the normal Cy5 FRET acceptor to observe arrival of a fluorescently labeled tRNA with a concomitant transition of the ribosome from the locked to the unlocked conformation. Our results illustrate the potential of nonfluorescent quenchers in single-molecule correlation studies.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Base Sequence
  • Carbocyanines / chemistry
  • Escherichia coli / genetics
  • Fluorescence Resonance Energy Transfer / methods*
  • Molecular Conformation*
  • RNA, Transfer / chemistry
  • RNA, Transfer / genetics
  • Ribosome Subunits, Large, Bacterial / chemistry
  • Ribosome Subunits, Large, Bacterial / genetics

Substances

  • Carbocyanines
  • cyanine dye 5
  • RNA, Transfer