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1krj

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{{STRUCTURE_1krj| PDB=1krj | SCENE= }}
{{STRUCTURE_1krj| PDB=1krj | SCENE= }}
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'''Engineering Calcium-binding site into Cytochrome c Peroxidase (CcP)'''
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===Engineering Calcium-binding site into Cytochrome c Peroxidase (CcP)===
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==Overview==
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We have previously shown that the K(+) site found in ascorbate peroxidase can be successfully engineered into the closely homologous peroxidase, cytochrome c peroxidase (CCP) (Bonagura, C. A. , Sundaramoorthy, M., Pappa, H. S., Patterson, W. R., and Poulos, T. L. (1996) Biochemistry 35, 6107-6115; Bonagura, C. A., Sundaramoorthy, M., Bhaskar, B., and Poulos, T. L. (1999) Biochemistry 38, 5538-5545). All other peroxidases bind Ca(2+) rather than K(+). Using the K(+)-binding CCP mutant (CCPK2) as a template protein, together with observations from structural modeling, mutants were designed that should bind Ca(2+) selectively. The crystal structure of the first generation mutant, CCPCA1, showed that a smaller cation, perhaps Na(+), is bound instead of Ca(2+). This is probably because the full eight-ligand coordination sphere did not form owing to a local disordering of one of the essential cation ligands. Based on these observations, a second mutant, CCPCA2, was designed. The crystal structure showed Ca(2+) binding in the CCPCA2 mutant and a well ordered cation-binding loop with the full complement of eight protein to cation ligands. Because cation binding to the engineered loop results in diminished CCP activity and destabilization of the essential Trp(191) radical as measured by EPR spectroscopy, these measurements can be used as sensitive methods for determining cation-binding selectivity. Both activity and EPR titration studies show that CCPCA2 binds Ca(2+) more effectively than K(+), demonstrating that an iterative protein engineering-based approach is important in switching protein cation selectivity.
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==About this Structure==
==About this Structure==
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[[Category: Pentagonal bipyrimidal geometry]]
[[Category: Pentagonal bipyrimidal geometry]]
[[Category: Trp191 cationic-radical]]
[[Category: Trp191 cationic-radical]]
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''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Wed Jul 2 10:50:30 2008''

Revision as of 07:50, 2 July 2008

Image:1krj.png

Template:STRUCTURE 1krj

Engineering Calcium-binding site into Cytochrome c Peroxidase (CcP)

Template:ABSTRACT PUBMED 10608846

About this Structure

1KRJ is a Single protein structure of sequence from Saccharomyces cerevisiae. Full crystallographic information is available from OCA.

Reference

Conversion of an engineered potassium-binding site into a calcium-selective site in cytochrome c peroxidase., Bonagura CA, Bhaskar B, Sundaramoorthy M, Poulos TL, J Biol Chem. 1999 Dec 31;274(53):37827-33. PMID:10608846

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