Förster resonance energy transfer

FRET Jablonski diagram

Förster Resonance Energy Transfer (FRET) is a mechanism describing energy transfer between two light-sensitive molecules. A donor molecule, excited by electromagnetic radiation, may transfer its excitation energy to an acceptor molecule through nonradiative means. This process is highly dependent on the distance between the donor and acceptor, making FRET a powerful tool in molecular biology for studying interactions at the nanoscale.

Overview

FRET occurs when two chromophores, a donor and an acceptor, are in close proximity to each other. The efficiency of energy transfer is inversely proportional to the sixth power of the distance between the donor and acceptor, a relationship described by the Förster equation. This sensitivity to distance makes FRET an invaluable technique for measuring distances within molecules or complexes in the range of 1 to 10 nanometers.

Mechanism

The underlying principle of FRET is based on the dipole-dipole coupling between the donor and acceptor molecules. When the donor molecule absorbs light, it reaches an excited state. If an acceptor molecule with an appropriate energy level is nearby, the excited state energy can be transferred to the acceptor through nonradiative means, often resulting in the emission of light from the acceptor molecule at a longer wavelength than the light absorbed by the donor.

Applications

FRET is widely used in biochemistry, molecular biology, and biophysics for various applications, including:

• Studying protein-protein interactions
• Monitoring conformational changes in proteins
• Measuring distances within or between biomolecules
• Investigating the dynamics of biological processes in living cells

Requirements

For FRET to occur, several conditions must be met:

• The emission spectrum of the donor must overlap with the absorption spectrum of the acceptor.
• The donor and acceptor must be within 1 to 10 nanometers of each other.
• The transition dipole moments of the donor and acceptor must be properly oriented.

FRET Efficiency

The efficiency of FRET can be quantified and is a crucial parameter for interpreting FRET experiments. It is defined as the fraction of the energy absorbed by the donor that is transferred to the acceptor. This efficiency is dependent on the distance between the donor and acceptor, the spectral overlap of the donor emission and acceptor absorption, and the relative orientation of the donor and acceptor dipoles.

Experimental Techniques

Several techniques are used to measure FRET efficiency, including:

• Fluorescence spectroscopy
• Fluorescence lifetime imaging microscopy (FLIM)
• Bioluminescence resonance energy transfer (BRET), a variant of FRET using bioluminescent proteins as donors

Limitations

While FRET is a powerful technique, it has limitations, including:

• The requirement for precise overlap between donor emission and acceptor absorption spectra
• Sensitivity to the orientation of donor and acceptor molecules
• The need for the donor and acceptor to be within a specific distance range

Conclusion

Förster Resonance Energy Transfer is a critical tool in the study of molecular interactions and dynamics. Its ability to provide insights into the nanoscale organization and movements of biomolecules makes it indispensable in the fields of molecular biology, biochemistry, and biophysics.

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