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O
H
HN
O
HO
O
R
NH
O
O
O
N
N
N
O
N
O
O
-O
(H / -)
N
R
R
NH
DsRed
GFP
O
O
O
O
N
N
O
N
O
N
-O
-O
N
N
OH
O
zFP538
mOrange
Fig. 1
Left
: VFPs share a universal “beta-can” structure of ~2.4 nm in diameter and 4.2 nm in
height. The light-emitting chromophore is formed in a multistep autocatalytic reaction within the
protein cylinder.
Right
: Schematic outlining chromophore formation in VFPs. The red-emitting
chromophore in DsRed and the green-emitting chromophore in GFP are different end products in a
multistep chemical reaction [
39
,
40
]. The red-emitting chromophore is an intermediate in the
formation of the chromophores in zFP538 [
41
] and mOrange [
42
]
been identified [
45
-
48
]. While this possibility to modify the emission of a fluores-
cent protein by means of applied light adds to the spectral complexity and versatil-
ity of VFPs, the photoactivation and photoswitching properties have made these
proteins an important tool in a number of applications, including the revolutionary
super resolution microscopy techniques.
The detailed photophysics and spectral appearance of an emitter is further
dependent on its embedding matrix. By virtue of its embedding within the protein
scaffold, the chromophore interacts with its local nanoenvironment defined by
the surrounding protein. The exact nature of the nanoenvironment of the chro-
mophorecanbemodifiedbymutatingsurrounding amino acid residues, resulting
in changes in the photophysical properties of the chromophore [
42
,
49
-
52
].
Although VFPs do not show major structural rearrangements as seen in many
enzymes, they are not static structures, so that thermally driven or photoinduced
reorientations in the chromophore vicinity can lead to a modification of photo-
physical parameters [
29
,
30
,
51
].
Finally, the tendency of many VFPs to form dimers and tetramers [
17
,
53
,
54
]
can lead to complex energy transfer interactions within the specific oligomeric
species formed.
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