One-Photon and Two-Photon Excitation of Fluorescent Proteins - Fluorescent Proteins: From Understanding to Design - page 6

excited state (few ns) is too short to allow equilibration, resulting in the observed
slight shift of emission peak (510-503 nm).

3 Chromophores of FPs

Chromophore structures of FPs and their formation mechanisms are summarized in
Fig. 4 . The range of excitation and emission wavelengths that they give rise to in
different representative FPs can be read in Table 1 .
In all natural FPs discovered so far, the prechromophore tripeptide has a
X 1 -Tyr 2 -Gly 3 sequence, where X 1 can be almost any amino acid. Chromophore
formation is inhibited upon mutation of the Gly 3 [ 13 ], suggesting that the peculiar
conformational flexibility of Glycine is necessary at that location. By contrast,
substitution of Tyr 2 with an aromatic amino acid (Phe, His, or Trp) preserves the
fluorescence. The resulting artificial mutants contain chromophores with the phenol
ring replaced by the corresponding aromatic ring, and have blueshifted excitation
and fluorescence wavelengths with respect to the parent protein. Blue FPs such as
EBFP [ 118 ], Azurite [ 22 ], and EBFP2 [ 21 ] all derive from Y66H av GFP (BFP
chromophore in Fig. 4 ) with additional mutations to improve folding efficiency,
brightness, and photostability. Y66W av GFP mutants (CFP in Fig. 4 ) such as
Cerulean [ 23 ] and ECFP [ 11 ] emit cyan light.
The other determinant of chromophore structure comes from modifications
around the X 1 a -carbon. With respect to the av GFP chromophore formation mech-

anism, an additional step occurs in DsRed [ 43 ] and other red fluorescent proteins
(RFPs), namely the oxidation of the C-N main-chain bond of X 1 , which leaves an
acylimine substituent at the corresponding position of the imidazolinone ring.
The sequence of events during RFP chromophore self-processing entails first the
oxidation leading to the acylimine substituent followed by dehydrogenation of
the bridging carbon [ 14 ]. The acylimine substituent enlarges the extension of the
p

-conjugated system, and correspondingly lowers the excitation energy, resulting
in red/orange fluorescence. Analogously to the case of the GFP chromophore,
substituting the Tyr 2 in this chromophore with Phe or Trp (RFP Y67F and RFP
Y67W in Fig. 4 ) results in blueshifted spectral properties, with fluorescence in the
blue (mBlueberry2 [ 21 ]) and orange (mHoneydew [ 32 ]) domain, respectively.
In eqFP611 and Rtms5, the RFP chromophore is in the trans (or E) isomer
instead of the cis (or Z) form normally present in other FPs [ 39 , 44 ]. It adopts a
coplanar conformation in eqFP611, whereas in Rtms5 it is highly non-coplanar.
This feature is linked with the very different quantum yields of the two proteins,
presumably because noncoplanar conformations favor non-radiative de-excitation
pathways [ 44 ].
Further reactions can take place around the acylimine moiety, such as side chain
cyclization by nucleophilic addition of the Thr (in mOrange), Cys (mKO), or Lys
(zFP538) side chain, the latter followed by backbone cleavage [ 45 - 47 ]. Backbone