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| - | [[Image:1ccs.gif|left|200px]] | + | {{Seed}} |
| | + | [[Image:1ccs.png|left|200px]] |
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| | {{STRUCTURE_1ccs| PDB=1ccs | SCENE= }} | | {{STRUCTURE_1ccs| PDB=1ccs | SCENE= }} |
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| - | '''STRUCTURE-ASSISTED REDESIGN OF A PROTEIN-ZINC BINDING SITE WITH FEMTOMOLAR AFFINITY'''
| + | ===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-->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.
| + | The line below this paragraph, {{ABSTRACT_PUBMED_7761440}}, adds the Publication Abstract to the page |
| | + | (as it appears on PubMed at http://www.pubmed.gov), where 7761440 is the PubMed ID number. |
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| | + | {{ABSTRACT_PUBMED_7761440}} |
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| | ==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.]] |
| - | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Fri May 2 12:35:21 2008'' | + | |
| | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Mon Jun 30 20:34:03 2008'' |
Revision as of 17:34, 30 June 2008
Template:STRUCTURE 1ccs
STRUCTURE-ASSISTED REDESIGN OF A PROTEIN-ZINC BINDING SITE WITH FEMTOMOLAR AFFINITY
Template:ABSTRACT PUBMED 7761440
About this Structure
1CCS 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
Page seeded by OCA on Mon Jun 30 20:34:03 2008
