intrinsic fluorescence of the protein. While this is a very convenient titration assay
when validated for an individual protein–ligand system, one has to be careful when
testing unknown pairs, because the same decrease in intensity can occur by collisional
quenching.
Highly effective quenchers for fluorescence emission are oxygen, as well as the
iodide ion. Usage of these quenchers allows surface mapping of biological macromol-
ecules. For instance, iodide can be used to determine whether tryptophan residues
are exposed to solvent.Fluorescence resonance energy transfer (FRET)
Fluorescence resonance energy transfer(FRET) was first described by Fo ̈rster in 1948.
The process can be explained in terms of quantum mechanics by a non-radiative energy
transfer from a donor to an acceptor chromophore. The requirements for this process are
a reasonable overlap of emission and excitation spectra of donor and acceptor chromo-
phores, close spatial vicinity of both chromophores (10–100 A ̊), and an almost parallel
arrangement of their transition dipoles. Of great practical importance is the correlationFRET/1
R^60 ð^12 :^6 Þshowing that the FRET effect is inversely proportional to the distance between donor
and acceptor chromophores,R 0.
The FRET effect is particularly suitable forbiological applications, since distances of
10–100 A ̊are in the order of the dimensions of biological macromolecules. Furthermore,
the relation between FRET and the distance allows for measurement of molecular distances
and makes this application a kind of ‘spectroscopic ruler’. If a process exhibits changes in
molecular distances, FRET can also be used to monitor the molecular mechanisms.
The high specificity of the FRET signal allows for monitoring of molecular interactions
and conformational changes with high spatial (1–10 nm) and temporal resolution (<1ns).
Especially the possibility of localising and monitoring cellular structures and proteins in
physiological environments makesthis method very attractive. The effects can be
observed even at low concentrations (as low as single molecules), in different environ-
ments (different solvents, including livingcells), and observations may be done in
real time.
In most cases, different chromophores are used as donor and acceptor, presenting two
possibilities to record FRET: either as donor-stimulated fluorescence emission of the
acceptor or as fluorescence quenching of the donor by the acceptor. However, the same
chromophore may be used as donor and acceptor simultaneously; in this case, the
depolarisation of fluorescence is the observed parameter. Since non-FRET stimulated
fluorescence emission by the acceptor can result in undesirable background fluores-
cence, a common approach is usage of non-fluorescent acceptor chromophores.
FRET-based assays may be used to elucidate the effects of new substrates for
different enzymes or putative agonists in a quick and quantitative manner. Further-
more, FRET detection might be used in high-throughput screenings (see Sections
17.3.2 and 18.2.3), which makes it very attractive for drug development.501 12.3 Fluorescence spectroscopy