Structure–Function Relationships in Fluorescent Marker Proteins of the Green Fluorescent Protein Family - Fluorescent Proteins: From Understanding to Design - page 246

3.5.2 Covalent Modifications of the Chromophore Structure

An alternative approach to color tuning involves modifying the structure of the
chromophore itself. For some species, additional chromophore modifications occur
spontaneously (autocatalytically) during folding and maturation of the protein
(see Sect. 2.3 ). Others require irradiation with light of suitable wavelengths and
thus are photochemical in nature (see Sect. 3.5.2). In addition, color tuning can be
achieved by varying the tripeptide composition using site-directed mutagenesis.

Site-Directed Mutagenesis

Substitution of Trp for Tyr at position 66 in the chromophore tripeptide produces a
chromophore with an indole instead of a phenol or phenolate moiety [ 73 ], with
excitation and emission wavelengths intermediate between those of the neutral
phenol and the anionic phenolate chromophores. However, the increased bulk of
the indole requires many additional mutations to regain a reasonable brightness of
these proteins [ 74 ], called cyan-fluorescent proteins, or CFPs, because of their
blue-green or cyan emission (Fig. 5 ). Substitution of His for Tyr shifts the emission
maximum to even shorter wavelengths than Trp66, generating the blue-fluorescent
proteins, BFPs (Fig. 5 )[ 73 ]. Introduction of the nonnatural amino acids O -methyl-
tyrosine and p -aminophenylalanine at position 66 resulted in blue-shifted, weak
fluorescence with respect to wild-type GFP [ 75 ]. Incorporation of the nonnatural
residues 3-amino-L-tyrosine and 3-fluoro-L-tyrosine into the chromophore of the
DsRed monomer caused a 12 nm red shift and a 12 nm blue shift in fluorescence
emission, respectively [ 76 ].
Verkhusha and coworkers generated a BFP with a neutral Tyr at position 66.
Assuming that the red chromophore of FPs such as TagRFP or mCherry is formed
from a blue intermediate, they used a rational design strategy to suppress formation
of the acylimine double bond specific to the red chromophore. It involved replacing
the first amino acid of the chromophore tripeptide Met65 by an aliphatic residue
such as Leu to reduce the polarization of the residue 65

C-H bond in the chromo-
phore [ 77 ]. The crystal structure of mTagBFP revealed that the chromophore is a
4-(4-hydroxybenzyl)-1H-imidazole-5-ol [ 78 ]. The C

a

a b

bond of Tyr66 is fully
reduced, and the hydroxyphenyl ring is rotated out of the chromophore plane.

-C

Fig. 5 Variations of and next to the chromophore-forming residue 66 in GFP: yellow -fluorescent
protein (YFP), GFP, cyan -fluorescent protein (CFP), blue -fluorescent protein (BFP)