1krj
From Proteopedia
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'''Engineering Calcium-binding site into Cytochrome c Peroxidase (CcP)''' | '''Engineering Calcium-binding site into Cytochrome c Peroxidase (CcP)''' | ||
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[[Category: Poulos, T L.]] | [[Category: Poulos, T L.]] | ||
[[Category: Sundaramoorthy, M.]] | [[Category: Sundaramoorthy, M.]] | ||
| - | [[Category: | + | [[Category: Bidentate bond]] |
| - | [[Category: | + | [[Category: Calcium selectivity]] |
| - | [[Category: | + | [[Category: Cation-binding loop]] |
| - | [[Category: | + | [[Category: Lignin peroxidase]] |
| - | [[Category: | + | [[Category: Open/closed conformer]] |
| - | [[Category: | + | [[Category: Pentagonal bipyrimidal geometry]] |
| - | [[Category: | + | [[Category: Trp191 cationic-radical]] |
| - | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Fri May 2 23:05:03 2008'' | |
| - | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on | + | |
Revision as of 20:05, 2 May 2008
Engineering Calcium-binding site into Cytochrome c Peroxidase (CcP)
Overview
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.
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 Page seeded by OCA on Fri May 2 23:05:03 2008
Categories: Cytochrome-c peroxidase | Saccharomyces cerevisiae | Single protein | Bhaskar, B. | Bonagura, C A. | Poulos, T L. | Sundaramoorthy, M. | Bidentate bond | Calcium selectivity | Cation-binding loop | Lignin peroxidase | Open/closed conformer | Pentagonal bipyrimidal geometry | Trp191 cationic-radical
