One-Photon and Two-Photon Excitation of Fluorescent Proteins - Fluorescent Proteins: From Understanding to Design - page 32

7 Outlook

The range of spectral properties displayed by FPs is surprisingly broad. The factors
determining such variety are the extension of the chromophore -electron system

and the interactions of the chromophore with the surrounding protein matrix. Using
mutagenesis on the already wide set of natural FPs, the researcher can play with
these two factors to achieve the desired absorption/emission characteristic. The
mechanisms of spectral tuning are understood in general terms. Nonetheless, a
predictive structure-property relationship has proven elusive. Due to the intercon-
nections among the determinants of optical properties, two mutations known to,
say, redshift DsRed fluorescence when included separately into the sequence may
not give rise to additional redshifting when included simultaneously. Properties
such as quantum yield or extinction coefficient are yet more unpredictable.
These difficulties have not prevented an array of remarkable accomplishments in
manipulating FP excitation/emission, from the early generation of blue- and red-
shifted variants of av GFP to more recent achievements of bright FPs in the near-
infrared region. Despite the number and the variety of FPs available, there is much
scope for advancement in further enlarging the available spectral window and
improving FP brightness.

p

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