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PDBsum entry 4jb8
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Hydrolase/de novo protein
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PDB id
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4jb8
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Contents |
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143 a.a.
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92 a.a.
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158 a.a.
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PDB id:
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| Name: |
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Hydrolase/de novo protein
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Title:
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Caspase-7 in complex with darpin c7_16
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Structure:
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Caspase-7 subunit p20. Chain: a. Engineered: yes. Caspase-7 subunit p11. Chain: b. Engineered: yes. Darpin c7_16. Chain: p. Engineered: yes
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Source:
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Homo sapiens. Human. Organism_taxid: 9606. Gene: casp7, mch3. Expressed in: escherichia coli. Expression_system_taxid: 562. Synthetic construct. Organism_taxid: 32630. Expression_system_taxid: 562
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Resolution:
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1.70Å
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R-factor:
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0.173
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R-free:
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0.194
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Authors:
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A.Fluetsch,M.A.Seeger,M.G.Gruetter
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Key ref:
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M.A.Seeger
et al.
(2013).
Design, construction, and characterization of a second-generation DARP in library with reduced hydrophobicity.
Protein Sci,
22,
1239-1257.
PubMed id:
DOI:
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Date:
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19-Feb-13
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Release date:
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21-Aug-13
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PROCHECK
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Headers
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References
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P55210
(CASP7_HUMAN) -
Caspase-7 from Homo sapiens
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Seq:
Struc:
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303 a.a.
143 a.a.
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DOI no:
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Protein Sci
22:1239-1257
(2013)
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PubMed id:
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Design, construction, and characterization of a second-generation DARP in library with reduced hydrophobicity.
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M.A.Seeger,
R.Zbinden,
A.Flütsch,
P.G.Gutte,
S.Engeler,
H.Roschitzki-Voser,
M.G.Grütter.
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ABSTRACT
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Designed ankyrin repeat proteins (DARPins) are well-established binding
molecules based on a highly stable nonantibody scaffold. Building on 13 crystal
structures of DARPin-target complexes and stability measurements of DARPin
mutants, we have generated a new DARPin library containing an extended
randomized surface. To counteract the enrichment of unspecific hydrophobic
binders during selections against difficult targets containing hydrophobic
surfaces such as membrane proteins, the frequency of apolar residues at
diversified positions was drastically reduced and substituted by an increased
number of tyrosines. Ribosome display selections against two human caspases and
membrane transporter AcrB yielded highly enriched pools of unique and strong
DARPin binders which were mainly monomeric. We noted a prominent enrichment of
tryptophan residues during binder selections. A crystal structure of a
representative of this library in complex with caspase-7 visualizes the key
roles of both tryptophans and tyrosines in providing target contacts. These
aromatic and polar side chains thus substitute the apolar residues valine,
leucine, isoleucine, methionine, and phenylalanine of the original DARPins. Our
work describes biophysical and structural analyses required to extend existing
binder scaffolds and simplifies an existing protocol for the assembly of highly
diverse synthetic binder libraries.
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Links
