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decarboxylated species appears to be a minor species, and the majority of the sample
has the native mass, suggesting it could occur as a side-reaction or that photoconver-
sion is heterogeneous in nature [
40
].
Irreversible photoconversion was also reported for the A148G mutant of
asFP595 (KFP) [
60
-
62
]. It was subsequently speculated that light-induced decar-
boxylation of Glu215 in this protein destabilises the
trans
zwitter-ionic chromo-
phore, producing the fluorescent (0.45 quantum yield) neutral
trans
state [
63
,
64
].
3
Irreversible Green-to-Red Photoconversion in Related
Fluorescent Proteins
A distinct class of photoconvertable fluorescent proteins are the “green-to-red”
convertible proteins, including
Anthozoa
-derived fluorescent proteins and variants
having chromophores derived from the His-Tyr-Gly tripeptide. The mechanism of
photoconversion is fundamentally different from that found in avGFP and involves
covalent photochemical modification. In the dark, these proteins mature to fully
fold and form a chromophore up to the green fluorescent state. Subsequent illumi-
nation with UV or blue violet light results in their irreversible conversion to a red
fluorescent state. Members of this group are called Kaede [
65
], EosFP and mEosFP
[
66
,
67
], KikGR [
68
,
69
,
71
] and Dendra [
70
] (Fig.
11
).
Kaede is from the stony coral
Trachyphyllia geoffroyi
, which changes the
fluorescence from green at 518 nm to red at 582 nm upon irradiation with blue
light at 400 nm [
65
]. Its chromophore is derived from the His-Tyr-Gly tripeptide.
Another example in the same class of a photoconvertible “green-to-red” fluorescent
proteins is EosFP from the coral
Lobophyllia hemprichii
[
67
]. EosFP has very
similar spectroscopic and photoconversion characteristics as “Kaede”.
Photoconversion of EosFP was shown to be initiated from the protonated
green form of the chromophore [
66
]. The X-ray structures of the green and the
red forms showed that cleavage occurred between His-62 N and C atoms[
66
], in
agreement with the proposal based on mass spectrometry and NMR spectros-
copy of peptides derived from photoconverted Kaede [
72
]. A model for the light-
driven
-elimination reactions that causes reddening in this class of fluorescent
proteins includes excited state chemistry that is proposed to be initiated by a
ESPT reaction [
66
,
71
,
72
](Fig.
12
). Time-resolved spectroscopic evidence for
the existence of an ultrafast ESPT reaction in Kaede or EosFP is not yet available,
but the Stokes shift of fluorescence is similar to that of the neutral GFP
A
state of
avGFP. It is proposed that the resonance structures in the deprotonated excited
state of the chromophore then lead to an intermediate with a cleaved N-C bond.
A number of different reaction models, involving different intermediates, have
been proposed so far for the green-to-red photoconversion. Mizuno et al. [
72
]
proposed that His62 in Kaede could become doubly protonated on the imidazole
ring, leading to backbone cleavage via
b
-elimination [
72
] Nienhaus et al. [
66
]
proposed an ESPT from the phenolic oxygen of the chromophore to the N
b
e
of
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