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PDBsum entry 5aao
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Fluorescent protein
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PDB id
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5aao
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PDB id:
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| Name: |
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Fluorescent protein
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Title:
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Crystal structure of fluorogen-activating designed ankyrin repeat protein (darpin) dimer in complex with malachite green
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Structure:
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Fad3210. Chain: a, b, c, d, e, f, g, h, i, j, k, l. Engineered: yes
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Source:
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Synthetic construct. Organism_taxid: 32630. Expressed in: escherichia coli. Expression_system_taxid: 83333. Expression_system_variant: xl1-blue
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Resolution:
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2.60Å
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R-factor:
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0.216
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R-free:
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0.262
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Authors:
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A.Batyuk,M.Schuetz,L.Kummer,Y.Wu,P.Mittl,A.Plueckthun
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Key ref:
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M.Schütz
et al.
(2016).
Generation of Fluorogen-Activating Designed Ankyrin Repeat Proteins (FADAs) as Versatile Sensor Tools.
J Mol Biol,
428,
1272-1289.
PubMed id:
DOI:
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Date:
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27-Jul-15
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Release date:
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03-Feb-16
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PROCHECK
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Headers
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References
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No UniProt id for this chain
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Key: |
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Secondary structure |
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CATH domain |
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DOI no:
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J Mol Biol
428:1272-1289
(2016)
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PubMed id:
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Generation of Fluorogen-Activating Designed Ankyrin Repeat Proteins (FADAs) as Versatile Sensor Tools.
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M.Schütz,
A.Batyuk,
C.Klenk,
L.Kummer,
S.de Picciotto,
B.Gülbakan,
Y.Wu,
G.A.Newby,
F.Zosel,
J.Schöppe,
E.Sedlák,
P.R.Mittl,
R.Zenobi,
K.D.Wittrup,
A.Plückthun.
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ABSTRACT
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Fluorescent probes constitute a valuable toolbox to address a variety of
biological questions and they have become irreplaceable for imaging methods.
Commonly, such probes consist of fluorescent proteins or small organic
fluorophores coupled to biological molecules of interest. Recently, a novel
class of fluorescence-based probes, fluorogen-activating proteins (FAPs), has
been reported. These binding proteins are based on antibody single-chain
variable fragments and activate fluorogenic dyes, which only become fluorescent
upon activation and do not fluoresce when free in solution. Here we present a
novel class of fluorogen activators, termed FADAs, based on the very robust
designed ankyrin repeat protein scaffold, which also readily folds in the
reducing environment of the cytoplasm. The FADA generated in this study was
obtained by combined selections with ribosome display and yeast surface display.
It enhances the fluorescence of malachite green (MG) dyes by a factor of more
than 11,000 and thus activates MG to a similar extent as FAPs based on
single-chain variable fragments. As shown by structure determination and in
vitro measurements, this FADA was evolved to form a homodimer for the activation
of MG dyes. Exploiting the favorable properties of the designed ankyrin repeat
protein scaffold, we created a FADA biosensor suitable for imaging of proteins
on the cell surface, as well as in the cytosol. Moreover, based on the
requirement of dimerization for strong fluorogen activation, a prototype FADA
biosensor for in situ detection of a target protein and protein-protein
interactions was developed. Therefore, FADAs are versatile fluorescent probes
that are easily produced and suitable for diverse applications and thus extend
the FAP technology.
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Links
