Skip to main content

Förster resonance energy transfer (FRET)

FRET is like a molecular ruler. It detects subtle, nano-scale changes in space. Combined with fast CCD and modern microscopic equipment, single molecule FRET (smFRET) is the perfect tool to probe both spacial and temporal domains of molecular interactions.

Fluorescence Microscopy is an essential tool for modern biological experimentation. It allows for non-ambiguous detection for molecules of interest while significantly improving the effective resolving power of diffraction limited optics. For our in vitro, single molecule experiment, we use total internal reflection microscopy (TIRF), which further eliminates background noises by exciting only a single layer on the sample substrate and is used for our smFRET studies.

Figure: TIRF Microscopy.
A) Our optical setup: 532 nm and 638 nm lasers are used for TIRF excitation. The image is collected through a 60X water immersion, 1.2 N.A. objective and then split by a Dualview optical splitter (645 nm dichroic mirror). The donor and acceptor signals then pass through optical filters (donor: 585/70 bandpass; acceptor: 655 longpass) before detection by an emCCD camera. B) Total internal reflection at the quartz:water interface produces an evanescent wave that excites only those molecules within ~200 nm of the slide surface.

[label type=”label-default”]
Image credit: Jake
[/label]

Förster resonance energy transfer (FRET), or more commonly (improperly) known as fluorescence resonance energy transfer (FRET), is a nonradiative, electric dipole-dipole interaction between two molecules that involves transfer of energy from one (called donor) to the other (called acceptor). In particular, single-molecule FRET (or smFRET) has emerged as a useful tool for detecting changes in the separation between two molecules of 10-100Å, a scale pertinent to molecular biological events.

FigureJablonski diagram of FRET with typical timescales indicated

[label type=”label-default”]
Image from Wikipedia
[/label]

The following figures illustrates the distance-FRET relationship and how a typical experiment looks like. In a FRET experiment, the donor is excited by laser, meaning that they gain energy. When a donor is close to an acceptor, FRET occurs, and the energy transfer efficiency increases as the distance between them decreases, resulting in a drop of donor’s energy (as indicated by its fluorescence intensity) and an increase of the acceptor’s energy (so that the acceptor, which was not excited, now is excited and becomes fluorescent). Different wavelength filters are used to distinguish the emissions from donors and acceptors, respectively. The FRET is then calculated from the donor intensity (time traces from a spot of interest, usually the location of a fluorescent molecule that’s tagged on the targets such as protein or DNA) and acceptor intensity.

Figure. FRET-Distance Relationship

[label type=”label-default”]
Image from German Cancer Research Center
[/label]

Figure. Donor-Acceptor fluorescence intensity time traces and resulting FRET traces.

[label type=”label-default”]
Image credit: Jake
[/label]

With smFRET, we are able to probe the following aspects of protein-DNA conformations and dynamics –

Protein-DNA Interactions:

  • Protein Stoichiometries
  • Dynamics of Protein Assembly
  • Complex Conformational Changes
    • Mobility of Proteins on DNA
    • Protein Conformational Changes

Protein-DNA Interactions:

  • Protein Stoichiometries
  • Dynamics of Protein Assembly
  • Complex Conformational Changes
    • Mobility of Proteins on DNA
    • Protein Conformational Changes

Our current and past studies using smFRET can be found below, or navigated through the ‘Single Molecule FRET’ category on the left sidebar.

  • Posts related to smFRET Studies

    • Combined AFM and Fluorescence Microscopy Technique
      AFM is a powerful technique for studying protein-protein and protein-DNA interactions. However, a significant limitation to AFM and other high-resolution microscopy is the difficulty of identifying different proteins in multi-protein complexes. To resolve this, we are interested in combining the ...
      More
    • Single Molecule FRET Studies of DNA Repair Dynamics – Overview
      We have developed single-molecule FRET methods to study conformational dynamics of DNA-protein complexes as well as to analyze large multi-protein complexes related to DNA repair. Motivation Our group focuses primarily on structure-functions studies of the DNA mismatch repair pathway. Although our group has ...
      More
    Read more