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PDBsum entry 6qw4
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Peptide binding protein
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PDB id
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6qw4
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References listed in PDB file
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Key reference
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Title
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The role of changing loop conformations in streptavidin versions engineered for high-Affinity binding of the strep-Tag ii peptide.
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Authors
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T.G.M.Schmidt,
A.Eichinger,
M.Schneider,
L.Bonet,
U.Carl,
D.Karthaus,
I.Theobald,
A.Skerra.
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Ref.
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J Mol Biol, 2021,
433,
166893-166893.
[DOI no: ]
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PubMed id
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Abstract
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The affinity system based on the artificial peptide ligand Strep-tag® II and
engineered tetrameric streptavidin, known as Strep-Tactin®, offers attractive
applications for the study of recombinant proteins, from detection and
purification to functional immobilization. To further improve binding of the
Strep-tag II to streptavidin we have subjected two protruding loops that shape
its ligand pocket for the peptide - instead of D-biotin recognized by the
natural protein - to iterative random mutagenesis. Sequence analyses of hits
from functional screening assays revealed several unexpected structural motifs,
such as a disulfide bridge at the base of one loop, replacement of the crucial
residue Trp120 by Gly and a two-residue deletion in the second loop. The mutant
m1-9 (dubbed Strep-Tactin XT) showed strongly enhanced affinity towards the
Strep-tag II, which was further boosted in case of the bivalent
Twin-Strep-tag®. Four representative streptavidin mutants were crystallized in
complex with the Strep-tag II peptide and their X-ray structures were solved at
high resolutions. In addition, the crystal structure of the complex between
Strep-Tactin XT and the Twin-Strep-tag was elucidated, indicating a bivalent
mode of binding and explaining the experimentally observed avidity effect. Our
study illustrates the structural plasticity of streptavidin as a scaffold for
ligand binding and reveals interaction modes that would have been difficult to
predict. As result, Strep-Tactin XT offers a convenient reagent for the
kinetically stable immobilization of recombinant proteins fused with the
Twin-Strep-tag. The possibility of reversibly dissociating such complexes simply
with D-biotin as a competing ligand enables functional studies in protein
science as well as cell biology.
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