Biology Reference
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cleavage also occurs in asFP595 [ 48 , 49 ]. 1 AsFP595 is normally nonfluorescent and
contains a trans (E) chromophore. Its fluorescent form is accessed either temporar-
ily or permanently by photoactivation (hence the alternative name kindling fluores-
cent protein, or KFP), and contains a cis (Z) chromophore.
A different mechanism of redshifting occurs in Kaede and related proteins (KikGR,
EosFP, Dendra), where UV excitation photoconverts the originally green-emitting
proteins (with a GFP-like chromophore) into RFPs by cleaving the backbone
between main-chain N and C
of the Histidine at X 1 . The cleavage is followed
by double-bond formation between C
a
a
and C
b
giving rise to the uvKaede chromo-
phore structure [ 34 , 35 ].
As in av GFP, chromophores with the phenol group exist as either neutral
(protonated phenol) or anionic (deprotonated phenol). The absorption of the neutral
form is always significantly blueshifted with respect to the anionic form. Changing
the buffer pH generally results in a fluorescence titration curve that reveals the
changing equilibrium between these two forms. Several FPs at physiological pH
contain only the anionic chromophore. av GFP and some of its mutants are excep-
tions to this general behavior, because they can present both states, existing in a
persistent equilibrium over a broad pH range.
It is worth mentioning that according to some computational investigations, in
some proteins, such as asFP595, the zwitterionic form (with the imidazolinone
nitrogen protonated) might be the emitting one [ 51 ]. In addition, the zwitterionic
and other protonation state such as the cationic form (protonated phenol and
imidazolinone) may be involved in FP photophysics [ 52 ]. The cation, however,
exists at too acidic conditions (pH
2) from studies of chromophore analogs in
solution [ 17 , 53 ], in which the protein is denatured. Hence, it is not considered
relevant to FPs optical response at physiological conditions.
<
4
Isolated Chromophores
Synthetic analogs of chromophores of FPs were the starting point for a variety of
experimental and computational studies aiming to examine all the factors influen-
cing optical properties of FPs. Isolated chromophore models in solution are poorly,
if at all, fluorescent at room temperature. However, they become fluorescent when
the temperature is lowered to 77 K [ 54 ]. Presumably the conformational freedom at
higher temperature enables non-radiative pathways of de-excitation.
The established experimental model for the GFP chromophore is a p -hydroxy-
benzylidene-2,3-dimethlimidazolone or p -HBDI (Table 2 ). Such a model encom-
passes the relevant
-conjugated system. It lacks, however, the side chain of X 1
(the first residue of the tripeptide). That p -HBDI is a fairly accurate model for the
p
1
AsFP595 chromophore structure has been subject to debate, regarding the possible presence of an
imino (NH) group in place of the keto group at position 2 of the imidazolinone (see discussion in
[ 50 ]).
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