Fluorescence Lifetime of Fluorescent Proteins - Fluorescent Proteins: From Understanding to Design - page 89

Owing to its tetrameric nature and its (incomplete) chromophore formation through
a green intermediate, lifetime experiments are performed which aimed at the
elucidation of intramolecular hetero-FRET [ 72 , 89 , 93 , 94 ]. Most experiments
show t Fl ¼

3.7 ns as longest lifetime component. It is again the question whether
energy migration can prolong t Fl . It appears, however, that monomerization and
subsequent conversion into the mFruit-family is predominantly detrimental to F Fl .
[ 95 - 99 ]. Hardly any lifetime experiments of either oligomeric or monomeric
proteins have been performed as tunable pulsed excitation sources are still
limited in this spectral range [ 6 , 100 ]. Comparative studies are vastly missing. In
the theoretical treatment, which only recently began [ 101 , 102 ], even more volume-
conserving mechanisms should be taken into account [ 103 ].
Some general statements can be made: the extinction coefficient of some
members of the mFruit-family exceeds a value of 10 5 M 1 cm 1 at the wavelength
of maximal absorbance [ 95 ], resulting in a reduction of t rad according to (2). The
red-shifted fluorescence of these FPs acts in the opposite direction owing to the n

3
dependence. As a first approximation, we expect that t rad of the new FPs is, hence,
similar to that of other FPs. The limited number of yet available experimental data,
however, points to a longer t rad ~ 5-11(!) ns [ 6 ]. The achievable F Fl and t Fl are not
higher than those of other FPs, which might be traced back to the increasing
importance of the energy-gap law (Fig. 4 ). Further comparative experiments,
which also consider the incomplete chromophore maturation, are strongly desired
for a better understanding.

4 Outlook

Long t Fl are an indication of high F Fl and are advantageous for most applications in
fluorescence technology. For avFPs, a good, but incomplete understanding of the
factors, which influence t Fl has been achieved in the past decade. On the one hand,
amino acid substitutions especially at positions 65, 145, 148, 203, 224 of the protein
sequence were found to influence t Fl . In some of these cases, satisfying explana-
tions how these mutations affect t Fl are lacking. On the other hand, comparative
structural investigations revealed beneficial protein conformations and pointed to
improvement strategies. The combination of both approaches by screening methods
appears promising in finding advanced proteins [ 61 ]. Whereas for CFPs and YFPs
proteins now exist where t Fl is close to the theoretical limit of t rad , more efforts
have to be spent for the design of correspondingly better BFPs and GFPs. Lifetime
experiments on these proteins, where broad distribution of t Fl showed up, indicate
that the aim of t Fl close to t rad is challenging but not hopeless. Indeed, mutations
might be introduced which favour stiffer protein conformations, thereby providing
an optimal geometry for a planar chromophore and a reduced lifetime heterogeneity
while maintaining other mandatory biophysical properties such as high expression
and maturation yields etc.