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1cct

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{{STRUCTURE_1cct| PDB=1cct | SCENE= }}
{{STRUCTURE_1cct| PDB=1cct | SCENE= }}
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'''STRUCTURE-ASSISTED REDESIGN OF A PROTEIN-ZINC BINDING SITE WITH FEMTOMOLAR AFFINITY'''
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===STRUCTURE-ASSISTED REDESIGN OF A PROTEIN-ZINC BINDING SITE WITH FEMTOMOLAR AFFINITY===
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==Overview==
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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--&gt;aspartate (T199D) and threonine-199--&gt;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--&gt;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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{{ABSTRACT_PUBMED_7761440}}
==About this Structure==
==About this Structure==
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[[Category: Christianson, D W.]]
[[Category: Christianson, D W.]]
[[Category: Ippolito, J A.]]
[[Category: Ippolito, J A.]]
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''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Mon Jun 30 20:34:01 2008''

Revision as of 17:34, 30 June 2008

Image:1cct.png

Template:STRUCTURE 1cct

STRUCTURE-ASSISTED REDESIGN OF A PROTEIN-ZINC BINDING SITE WITH FEMTOMOLAR AFFINITY

Template:ABSTRACT PUBMED 7761440

About this Structure

1CCT is a Single protein structure of sequence from Homo sapiens. Full crystallographic information is available from OCA.

Reference

Structure-assisted redesign of a protein-zinc-binding site with femtomolar affinity., Ippolito JA, Baird TT Jr, McGee SA, Christianson DW, Fierke CA, Proc Natl Acad Sci U S A. 1995 May 23;92(11):5017-21. PMID:7761440

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