Fluorescence Lifetime of Fluorescent Proteins - Fluorescent Proteins: From Understanding to Design - page 77

ESPT is in FPs certainly one of the most evident and well-investigated mechan-
isms. Reversible cis-trans-isomerization can also occur when chromophoric sys-
tems, such as the chromophores of all FPs, contain exocyclic double-bonds. These
isomerization reactions likely proceed via CI and are therefore ultrafast [ 38 ].
Photon-induced electron transfer (PET), in which transiently an electron is trans-
ferred between a nearby group and the chromophore, is hardly detectable if the
back-reaction is faster than the forward reaction. Its prominent role can be con-
cluded from the irreversible photoconversion of FPs. Here, PET can be deciphered
as mechanism due to the irreversible release of carbon dioxide [ 10 ]. Thus, PET
might generally show up in the photodynamics, e.g. in the fluorescence quenching
of FP chromophores in the anionic form by halide anions, but experimental evi-
dence is lacking. Theoretical calculations, however, struggle with the open-shell
systems of transiently unpaired electrons.
Predictable changes of t Fl can be made in F orster resonance energy transfer

(FRET). The good understanding of the theoretical background allows for design-
ing energy donor-acceptor pairs with a sensing unit in between. FRET can be read-
out spectrally by taking the ratio of the donor and acceptor fluorescence, but also by
lifetime changes of the donor [ 39 ]. The latter approach becomes especially
promising if the acceptor is not fluorescent. Furthermore, lifetime changes do not
depend on concentration changes or on fluctuations of the excitation intensity.
Indeed, we recently developed a sensor for Cu 2+ , where we exploited that Cu 2+
exhibits a strong blue colour, i.e., broad absorbance of yellowish light, when bound
to proteins [ 40 ].
Reduced t Fl can also result from aggregation. In the particular case of FPs,
energy transfer on the picosecond time scale in homo- or hetero-dimers and
oligomers distributes the energy among the individual chromophores (Sch

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uttrigkeit
et al. 2001) [ 20 ]. If one of the chromophores is in a dark state, then fluorescence
vanishes. Self-quenching due to the formation of excimers, which can occur for
organic dyes in close contact, is only a minor issue in FPs as the chromophores are
buried deep within the protein barrel.

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1.6 Importance of Long Fluorescence Lifetimes

The most important aspect of long t Fl is met when different FPs with chromophores
of similar electronic structure are compared. In this case, the measured fluorescent
lifetime is proportional to the fluorescent quantum yield F Fl (10). A long t Fl

therefore corresponds to a high F Fl .

F Fl ¼ t Fl A 21 : (10)

Most GFPs and YFPs actually fulfil these requirements of similar electronic
structures, and we verified this way of computing F Fl by comparison with a classical
approach [ 21 ]. High F Fl are beneficial in FRET approaches, in single-molecule