Synthetic Biology of Autofluorescent Proteins - Fluorescent Proteins: From Understanding to Design - page 123

microscopy. The only disadvantage of this system is the relatively broad emission
peak, whereas the quantum yield is similar to ECFP [ 70 ].
It should be mentioned again that most commonly used FPs usually have poor
brightness when illuminated with UV light

366 nm. In order to address this
general limitation, Skerra and co-workers [ 73 ] recently combined in a FRET system
the fluorescence activity of a FP with the spectrally matching fluorescence of a
NCAA translated into the protein during its expression. In particular, they suc-
ceeded in incorporating site-specifically (~20 ˚ away from the protein's fluoro-
phore) the fluorescent (7-hydroxycoumarin-4-yl-)ethylglycine in ECFP. They chose
ECFP as the FRET acceptor for this NCAA because its absorption maximum in the
range of 430-450 nm overlaps with the 7-hydroxycoumarin emission maximum
( l max approx. 450 nm). Expectedly, the ECFP modified in this way exhibited
efficient FRET between its two fluorophores, having 'cyan' emission at 476 nm
upon excitation in the near-UV at 365 nm.
Finally, new classes of autofluorescent proteins could be generated using
NCAA. For instance, we could recently demonstrate that a single Trp

<

(4-Aza)
Trp substitution turned colorless anxA5 into a blue fluorescent protein [ 74 ]. The
same effects are known for (6-Aza)Trp and (7-Aza)Trp but these chromophores
suffer either from lower biocompatibility or fluorescence intensity compared to
(4-Aza)Trp [ 75 ]. In the future, we will explore the utilities of designing autofluor-
escent proteins by using fluorescent amino acids as building blocks for protein
biosynthesis.

!

References

1. Sample V, Newman RH, Zhang J (2009) The structure and function of fluorescent proteins.
Chem Soc Rev 38(10):2852-2864
2. Stepanenko OV, Verkhusha VV, Kuznetsova IM, Uversky VN, Turoverov KK (2008) Fluo-
rescent proteins as biomarkers and biosensors: throwing color lights on molecular and cellular
processes. Curr Protein Pept Sci 9(4):338-369
3. Chattoraj M, King BA, Bublitz GU, Boxer SG (1996) Ultra-fast excited state dynamics in
green fluorescent protein: multiple states and proton transfer. Proc Natl Acad Sci USA 93
(16):8362-8367
4. Tsien RY (1998) The green fluorescent protein. Annu Rev Biochem 67:509-544
5. Crameri A, Whitehorn EA, Tate E, Stemmer WPC (1996) Improved green fluorescent protein
by molecular evolution using DNA shuffling. Nat Biotechnol 14(3):315-319
6. Cormack BP, Valdivia RH, Falkow S (1996) Facs-optimized mutants of the green fluorescent
protein (gfp). Gene 173(1):33-38
7. Heim R, Tsien RY (1996) Engineering green fluorescent protein for improved brightness,
longer wavelengths and fluorescence resonance energy transfer. Curr Biol 6(2):178-182
8. Ormo M, Cubitt AB, Kallio K, Gross LA, Tsien RY, Remington SJ (1996) Crystal structure of
the Aequorea victoria green fluorescent protein. Science 273(5280):1392-1395
9. Heim R, Prasher DC, Tsien RY (1994) Wavelength mutations and posttranslational autoxida-
tion of green fluorescent protein. Proc Natl Acad Sci USA 91(26):12501-12504
10. Matz MV, Fradkov AF, Labas YA, Savitsky AP, Zaraisky AG, Markelov ML, Lukyanov SA
(1999) Fluorescent proteins from nonbioluminescent anthozoa species. Nat Biotechnol 17(10):
969-973