FRET Spectroscopy Flashcards
Why is molecular oxygen relevant to FRET spectroscopy?
It is an efficient quencher, and exists in a triplet state while in it’s ground state. It is also able to promote non-spin conserving transitions.
What is the basic principle of FRET?
Measuring the efficiency of transfer and hence separation of an acceptor-donor pair.
How do FRET acceptors differ from normal quenchers?
Normal quenchers remain in their ground state throughout the reaction, FRET acceptors are excited by the reaction.
How is the energy transferred between the donor and acceptor?
Resonance Energy Transfer. The oscillating dipole in the donor induces an oscillating dipole in the acceptor. When the resonance condition is satisfied (energy gap between the two excited states and their ground states are similar) the energy is transferred.
What are the two expansions of the FRET acronym?
Fluorescence Resonance Energy Transfer and Forster Resonance Energy Transfer
What determines the Forster distance for a transfer?
The size of the overlap between the donor emission spectrum and the acceptor absorption spectrum.
What is the most likely scenario for use of FRET spectroscopy?
Between an acceptor and donor molecule connected by a network of covalent bonds, usually a protein chain.
What is the Forster Distance, Ro?
The distance at which half the energy is transferred.
How is the energy transfer mechanism in FRET often described?
As involving ‘virtual photons’, that transfer the energy quickly enough that they are never observed. On this basis it would not be classed as non-radiative.
What are some common biochemical uses of FRET?
Probing conformation changes in macromolecules, often by linking donors/acceptors to the N and C terminals of a protein.
