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PDBsum entry 1ccs
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Lyase (oxo-acid)
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PDB id
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1ccs
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References listed in PDB file
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Key reference
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Title
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Structure-Assisted redesign of a protein-Zinc-Binding site with femtomolar affinity.
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Authors
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J.A.Ippolito,
T.T.Baird,
S.A.Mcgee,
D.W.Christianson,
C.A.Fierke.
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Ref.
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Proc Natl Acad Sci U S A, 1995,
92,
5017-5021.
[DOI no: ]
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PubMed id
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Note In the PDB file this reference is
annotated as "TO BE PUBLISHED".
The citation details given above were identified by an automated
search of PubMed on title and author
names, giving a
percentage match of
88%.
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Abstract
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We have inserted a fourth protein ligand into the zinc coordination polyhedron
of carbonic anhydrase II (CAII) that increases metal affinity 200-fold (Kd = 20
fM). The three-dimensional structures of threonine-199-->aspartate (T199D) and
threonine-199-->glutamate (T199E) CAIIs, determined by x-ray crystallographic
methods to resolutions of 2.35 Angstrum and 2.2 Angstrum, respectively, reveal a
tetrahedral metal-binding site consisting of H94, H96, H119, and the engineered
carboxylate side chain, which displaces zinc-bound hydroxide. Although the
stereochemistry of neither engineered carboxylate-zinc interaction is comparable
to that found in naturally occurring protein zinc-binding sites, protein-zinc
affinity is enhanced in T199E CAII demonstrating that ligand-metal separation is
a significant determinant of carboxylate-zinc affinity. In contrast, the
three-dimensional structure of threonine-199-->histidine (T199H) CAII,
determined to 2.25-Angstrum resolution, indicates that the engineered imidazole
side chain rotates away from the metal and does not coordinate to zinc; this
results in a weaker zinc-binding site. All three of these substitutions nearly
obliterate CO2 hydrase activity, consistent with the role of zinc-bound
hydroxide as catalytic nucleophile. The engineering of an additional protein
ligand represents a general approach for increasing protein-metal affinity if
the side chain can adopt a reasonable conformation and achieve inner-sphere zinc
coordination. Moreover, this structure-assisted design approach may be effective
in the development of high-sensitivity metal ion biosensors.
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