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PDBsum entry 3kcs
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Fluorescent protein
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PDB id
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3kcs
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References listed in PDB file
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Key reference
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Title
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Photoactivation mechanism of pamcherry based on crystal structures of the protein in the dark and fluorescent states.
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Authors
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F.V.Subach,
V.N.Malashkevich,
W.D.Zencheck,
H.Xiao,
G.S.Filonov,
S.C.Almo,
V.V.Verkhusha.
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Ref.
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Proc Natl Acad Sci U S A, 2009,
106,
21097-21102.
[DOI no: ]
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PubMed id
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Abstract
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Photoactivatable fluorescent proteins (PAFPs) are required for super-resolution
imaging of live cells. Recently, the first red PAFP, PAmCherry1, was reported,
which complements the photo-activatable GFP by providing a red super-resolution
color. PAmCherry1 is originally "dark" but exhibits red fluorescence after
UV-violet light irradiation. To define the structural basis of PAmCherry1
photoactivation, we determined its crystal structure in the dark and red
fluorescent states at 1.50 A and 1.65 A, respectively. The non-coplanar
structure of the chromophore in the dark PAmChery1 suggests the presence of an
N-acylimine functionality and a single non-oxidized C(alpha)-C(beta) bond in the
Tyr-67 side chain in the cyclized Met-66-Tyr-67-Gly-68 tripeptide. MS data of
the chromophore-bearing peptide indicates the loss of 20 Da upon maturation,
whereas tandem MS reveals the C(alpha)-N bond in Met-66 is oxidized. These data
indicate that PAmCherry1 in the dark state possesses the chromophore
N-[(E)-(5-hydroxy-1H-imidazol-2-yl)methylidene]acetamide, which, to our
knowledge, has not been previously observed in PAFPs. The photoactivated
PAmCherry1 exhibits a non-coplanar anionic DsRed-like chromophore but in the
trans configuration. Based on the crystallographic analysis, MS data, and
biochemical analysis of the PAmCherry1 mutants, we propose the detailed
photoactivation mechanism. In this mechanism, the excited-state PAmCherry1
chromophore acts as the oxidant to release CO(2) molecule from Glu-215 via a
Koble-like radical reaction. The Glu-215 decarboxylation directs the carbanion
formation resulting in the oxidation of the Tyr-67 C(alpha)-C(beta) bond. The
double bond extends the pi-conjugation between the phenolic ring of Tyr-67, the
imidazolone, and the N-acylimine, resulting in the red fluorescent chromophore.
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Figure 2.
Structures of the PAmCherry1 chromophore within its
environment in the OFF (A) and ON (B) states. Water molecules
are shown as red spheres; hydrogen bonds are shown as green
dashed lines. The mCherry chromophore and its environment (PDB
ID code 2H5Q) are shown for comparison (C).
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Figure 3.
Suggested mechanisms for the formation of the PAmCherry1 dark
chromophore (OFF state) and its light-induced conversion into
the fluorescent state (ON state) are shown. The cyclized form is
the chromophore with the non-oxidized bond between
p-hydroxyphenyl and imidazolone moieties and without
N-acylimine. Hydrogen bonds are shown with dashed lines.
Intermediate compounds are shown in parentheses. The chromophore
in the excited state is denoted with asterisk. hv, indicates the
illumination with violet light. Migration of the electron
density is shown with curved arrows.
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