paGFP) [ 10 ], the protonation equilibrium of p-HBI can be shifted irreversibly from
the neutral to the anionic species by intense light irradiation, causing decarboxyl-
ation of Glu212 [ 61 ]. Stabilization of the neutral form, by contrast, is a strategy to
generate blue-fluorescent variants (in the absence of ESPT) [ 62 ].
Blue-Shifted Chromophore Bands
Polar interactions of the p -HBI chromophore with residues in its vicinity may
reduce the extent of charge delocalization in the chromophore, resulting in hypso-
chromic band shifts of the anionic chromophore bands to the blue and cyan regions
of the spectrum. This effect explains the blue shift of the absorption and emission of
Dendra2 with respect to the very similar EosFP protein [ 63 ]. A comparison of the
X-ray structures revealed a single structural difference close to the chromophore. In
EosFP, the N
1 atom of Arg66 forms a hydrogen bond to the carbonyl oxygen of
the imidazolinone. In contrast, the Arg66 side chain in Dendra2 is pulled away from
the imidazolinone ring, held in place by, among other interactions, a weak hydrogen
bond between the Arg66 N
and the hydroxyl side chain of Thr69, resulting in
reduced negative charge stabilization on the imidazolinone ring. Electron density is
known to shift from the phenyl to the imidazolinone upon electronic excitation of
the p -HBI chromophore, so that the missing charge stabilization on the imidazoli-
none by Arg66 in Dendra raises the energy of the excited state with respect to the
ground state, leading to a blue shift of the transition. In complete agreement with
this explanation, the Thr69Ala mutation turns Dendra2 into a more EosFP-like
protein, whereas the Ala69Thr mutation of EosFP elicits the opposite effect [ 63 ].
A threonine residue at position 69 has been found in a number of blue or cyan FPs,
suggesting a general role of this blue-shifting mechanism (Table 3 in [ 63 ]).
In another coral FP, anobCFP, Glu167 is involved in the blue-shifted fluores-
cence, as its replacement by glycine shifts the emission maximum by 10 nm to the
red [ 35 ]. Because this mutation also leads to the disappearance of the 425-nm
excitation peak, Glu167 most likely stabilizes the neutral form of the chromophore.
mKate is a far-red FP emitting at 635 nm [ 64 ]. By replacing Met167 in close vicinity
to the chromophore by either Glu or Asp, a hydrogen bond to the hydroxyphenyl is
established. Because the p K a values of Glu and Asp are expected to be lower than
that of the chromophore, these mutations stabilize the neutral chromophore, which
has an absorption peak at ~460 nm [ 65 ]. Upon excitation, the chromophore p K a
drops, resulting in ESPT from the hydroxyphenyl to residue 167, and red emission
occurs. The blue-shift in the red emission maximum with respect to mKate reflects
the stabilization of the negative charge on the hydroxyphenyl ring.
Red-Shifted Chromophore Bands
The chromophore environment may also induce bathochromic shifts of the fluores-
cence to the yellow or red region of the spectrum. For example, in the YFP variant