One-Photon and Two-Photon Excitation of Fluorescent Proteins - Fluorescent Proteins: From Understanding to Design - page 23

However, since in av GFP and its green variants, the chromophore is still involved in
relevant interactions with surrounding residues and water molecules, a more accu-
rate picture is that of a cancellation of the various contributions. Computational
studies [ 85 ] point at a counteraction between the redshifting H-bond with Arg at
av 96 (a conserved residue in FPs, being a requisite for chromophore formation) and
the blue shifting of H-bonds to the phenolate. Slow-maturing av GFP variants with
mutated Arg96 do exist, and also shed light on the relevance of Arg96 on the optical
properties. Getzoff and coworkers [ 90 ] structurally solved the mature R96M mutant,
where the H-bond with the imidazolinone carbonyl group is abolished, and replaced
with a, somewhat weaker, H-bond with a water molecule (donor-acceptor distance is
2.9 ˚ in PDB structure 2AWK). In line with theoretical predictions [ 85 ], the R96M
mutant displays blueshifted optical properties (Table 5 ). Its application as a cyan-
emitting protein is, however, severely hampered by the extremely slow chromophore
maturation (months).

5.1.3 Yellow Group

The common feature of proteins in the yellow group is the Tyr or Phe at av 203 (see
Fig. 8 ). The T203Y mutation was introduced in av GFP mutants with the rationale
that an aromatic amino acid at that position would be

-stacked to the chromophore
phenolate, thereby lowering the excitation energy by increasing the polarizability of
the environment around the chromophore [ 27 ]. The mutation indeed resulted in
redshifted variants (see Table 5 ), and X-ray studies confirmed the

p

stacking
configuration [ 27 ]. Notably, the same structural stratagem for redshifting is pre-
sumably at play in a natural FP named phiYFP [ 30 ], also featuring a Tyr at av 203.
One additional important feature arose from T203Y mutation, i.e., a rather
enhanced extinction coefficient (see Table 1 ). Similar spectral shifts are obtained
with a phenylalanine replacement in the av GFP T203F mutant [ 97 ]. Actually, the
absorption/excitation red shift in these Thr203 mutants is only partially accounted for
by the

p

-stacking. Indeed, also T203V and T203I mutants show a redshifted absorp-
tion with respect to av GFP (from 477 nm of av GFP to 502 of the T203V and T203I
mutants [ 97 ]), associated with the removed Thr203 H-bond to the phenolate. Red-
shifted fluorescence is instead almost totally ascribed to the

p

-stacked Tyr or Phe.
The case of CpYGFP is worth describing. CpYGFP features a Histidine at av 63,
which is stacked above the chromophore (i.e., on the opposite side with respect to
Y203 in the orientation of Fig. 8 ) and presumably neutral, giving rise to rather
redshifted excitation/emission [ 96 ]. Replacement of this His with Thr (the residue
present in the equivalent position in av GFP) blueshifts the excitation/emission to
495/511 nm. Instead, replacement with Phe additionally redshifts to 514/522 nm, as
a consequence of a larger amount of

p

-stacking.
Both EYFP (enhanced YFP) and CpYGFP X-ray structures show a peculiar
H-bond between Glu at av 222 and the imidazolinone of the chromophore. It would
be tempting to assign a role in redshifting to this H-bond. Although X-ray structures
of other yellow-emitting av GFP variants such as E 2 GFP [ 19 ] (PDB Code 2H9W)

p