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properties prevented researchers from thorough investigations [ 66 ]. The intro-
duction of H66 yields a protein with an averaged t Fl <
1 ns at room temperature
[ 67 , 68 ]. Already, the additional mutations Y145F and S65T lead to proteins with
distinctly prolonged t Fl , which could be further increased up to 3.65 ns by screening
methods [ 49 , 51 , 64 , 66 ]. Here, especially the mutation V224R appears beneficial
for a long t Fl . A reduced conformational freedom or a stiffer hydrogen-bonding
network is made responsible [ 69 ]. In combination with F Fl ~ 0.5, one could argue
on the basis of (9) that the radiative lifetime t rad should be around 7 ns. However, all
time-resolved experiments on BFPs have shown so far multiexponential decay
behaviour for t Fl which suggests a strong heterogeneity of the chromophore sur-
rounding. According to this model, some protein conformations lock the conjugated
system in weakly or non-fluorescent states where rotations in the excited-state
around the exocyclic C-C bonds are facilitated. These movements appear to be
thermally activated and low-temperature experiments indeed show that the short
lifetime components vanish [ 51 , 67 ]. It should be noted that the longer lifetime
component, despite a distinct temperature dependence, levels off at a maximum
value t Fl ~ 3.5 ns, which appears to be t rad in cryo-protectants.
Another way of disabling rotations around the exocyclic double bonds is the coor-
dination of metal ions by the nitrogen-atoms of the imidazolinone and the histidine.
It would be interesting to see whether binding of Zn 2+ , which is not redox active and
which cannot act as FRET quencher, to both heterocycles of the chromophore would
result in similar values of t Fl at room and at low temperatures [ 70 ].
If we define BFP just by its fluorescence colour, then two further types of
proteins should be mentioned: First, Y66F variants which exhibit t Fl <<
room temperature but t rad ~ 4.5 ns at cryogenic temperatures [ 67 ]; second, proteins
containing Y66 where the ESPT is suppressed. This effect was observed in several
proteins containing the mutation E222Q, and t Fl up to 1.5 ns at room temperature
was observed [ 21 ]. However, further prolongation of t Fl and systematic investiga-
tions are missing yet owing to the low expression yields of E222Q containing
proteins. In an alternative approach, suppression of ESPT was be achieved by the
mutations S205V/T203V in enhanced GFP (eGFP) leading to the bright BFP
mKalama [ 71 ]. In the same work, other bright BFPs, partially derived from other
coral proteins, were presented which all exhibit F Fl ~ 0.5.
3.2 Cyan Fluorescent Proteins
Cyan Fluorescent Proteins (CFPs), where the central amino acid of the chromo-
phore triad is replaced by tryptophane [ 63 ] (Fig. 7 , right), were disregarded in
terms of its photophysical characterization for almost one decade. The fluorescence
maximum below 500 nm overlaps with strong cellular autofluorescence, and there-
fore, application of CFPs is mostly restricted to its use as FRET-donor [ 72 ]. A high-
resolution crystallographic analysis of eCFP initiated further improvements: two
different conformations are found and are correlated with two different lifetimes as
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