Fluorescence Lifetime of Fluorescent Proteins - Fluorescent Proteins: From Understanding to Design - page 83

3 Fluorescence Lifetime of FPs
Most attempts to improve FPs were focused so far on fluorescence intensities, the
spectral characteristics and the factors which have a strong impact on them.
Fluorescence lifetimes were measured, if at all, as just one parameter among
other spectroscopic data. It is therefore not astonishing that the values for t Fl ,
provided by different research groups, frequently vary more than the stated accu-
racy of the respective analysis. One prototypic example is t Fl of Cerulean, a CFP
variant. It finds application as improved donor in FRET-constructs. Published
values of t Fl span the range from 2.9 to 3.8 ns (Hoffmann et al. 2008) [ 58 , 61 , 62 ].
It should be noted that similar examples are widespread and can be found for YFPs
as well as for dsRed. These ubiquitous deviations can be, at least in part, due to the
alterations (temperature, refractive index) mentioned in the preceding sections;
they could also hint at hidden quenchers or even at the proteins' history. Finally,
even the mathematical treatment, i.e., monoexponential vs. biexponential fitting
vs. fitting with a distribution of lifetimes can influence the outcome [ 59 ]. Hence,
we prefer systematic investigations, where the effects of mutations are compared
and from which qualitative conclusions are drawn.
3.1 Blue Fluorescent Proteins
The so-called blue fluorescent proteins (BFPs) denote FPs where the central amino
acid Y66 in the wild-type GFP of the jellyfish Aequorea victoria is replaced by
histidine (Y66H) [ 63 ] (Fig. 7 , left). Only few time-resolved experiments were
performed so far on this family, and it seems that the low F Fl and other detrimental
Fig. 7 Blue and cyan fluorescent proteins. BFPs are, classically, proteins with Y66H but could
also be established if ESPT is disabled in Y66-containing proteins [ 21 , 64 ]. CFPs contain the
mutation Y66W. For the optimized CFP variant Cerulean, an alternative conformation was found
at low pH which apparently exhibits a smaller t Fl [ 58 , 59 , 65 ]