Molecular spectroscopy and structure: FRET Flashcards
How can we quantify how non-radiative processes reduce the efficiency of radiative processes?
using quantum yields and simple rate equations and determining the rate of quenching using a Stern-Volmer plot
Give some examples of efficient quenchers
bromide - bromide ions have states at very similar energies to the excited states of many organic molecules
oxygen
Why is oxygen peculiar?
it exists in a triplet state in its ground state and is able to promote non-spin-conserving transitions
When is the resonance condition satisfied for D and A?
when the energy gap between D(S0) and D(S1)* is similar to that between the ground and excited states of A
Outline the general scheme under which this could occur
D (S0) + hv —> D* (S1)
D* (S1) + A (S0) —> A* (S1)
A*(S1) —> A (S0) + hv’
What does FRET stand for?
fluorescence resonance energy transfer or Forster resonance energy transfer
When can FRET occur?
- when D and A are separated by a short distance (
What does the efficiency of energy transfer from D to A depend on?
n(T) depends strongly on the distance R between donor and acceptor
What equation links n(T) and R?
n(T) = (R0)^6/[ R^6 + (R0)^6 ]
Which linearised equation links n(T) and R?
1/n(T) = 1 + (R/R0)^6
What is the Forster distance?
the distance at which half of the energy is transferred
How is n(T) determined?
by comparing the fluorescence quantum yield of the donor molecule in the absence of the acceptor molecule with the fluorescence quantum yield of the donor once the acceptor is present
How can changes in conformation of biomolecules be investigated?
by placing two genetically encoded dyes onto the N- and C-termini of myosin, one a donor and the other an acceptor, to observe any changes in efficiency of the FRET and hence changes in conformation
Why is FRET referred to as a non-radiative process?
because the ‘virtual photons’ involved are never observed
When is FRET used in biochemistry?
to determine proximity, spacing and shape of various species
