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| - | [[Image:1krj.gif|left|200px]] | |
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| - | {{Structure
| + | ==Engineering Calcium-binding site into Cytochrome c Peroxidase (CcP)== |
| - | |PDB= 1krj |SIZE=350|CAPTION= <scene name='initialview01'>1krj</scene>, resolution 2.00Å
| + | <StructureSection load='1krj' size='340' side='right'caption='[[1krj]], [[Resolution|resolution]] 2.00Å' scene=''> |
| - | |SITE=
| + | == Structural highlights == |
| - | |LIGAND= <scene name='pdbligand=HEM:PROTOPORPHYRIN+IX+CONTAINING+FE'>HEM</scene>, <scene name='pdbligand=K:POTASSIUM+ION'>K</scene> | + | <table><tr><td colspan='2'>[[1krj]] is a 1 chain structure with sequence from [https://en.wikipedia.org/wiki/Saccharomyces_cerevisiae Saccharomyces cerevisiae]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1KRJ OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=1KRJ FirstGlance]. <br> |
| - | |ACTIVITY= <span class='plainlinks'>[http://en.wikipedia.org/wiki/Cytochrome-c_peroxidase Cytochrome-c peroxidase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=1.11.1.5 1.11.1.5] </span>
| + | </td></tr><tr id='method'><td class="sblockLbl"><b>[[Empirical_models|Method:]]</b></td><td class="sblockDat" id="methodDat">X-ray diffraction, [[Resolution|Resolution]] 2Å</td></tr> |
| - | |GENE= OPBYC ([http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&srchmode=5&id=4932 Saccharomyces cerevisiae])
| + | <tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat" id="ligandDat"><scene name='pdbligand=HEM:PROTOPORPHYRIN+IX+CONTAINING+FE'>HEM</scene>, <scene name='pdbligand=K:POTASSIUM+ION'>K</scene></td></tr> |
| - | |DOMAIN=
| + | <tr id='resources'><td class="sblockLbl"><b>Resources:</b></td><td class="sblockDat"><span class='plainlinks'>[https://proteopedia.org/fgij/fg.htm?mol=1krj FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=1krj OCA], [https://pdbe.org/1krj PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=1krj RCSB], [https://www.ebi.ac.uk/pdbsum/1krj PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=1krj ProSAT]</span></td></tr> |
| - | |RELATEDENTRY= | + | </table> |
| - | |RESOURCES=<span class='plainlinks'>[http://oca.weizmann.ac.il/oca-docs/fgij/fg.htm?mol=1krj FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=1krj OCA], [http://www.ebi.ac.uk/pdbsum/1krj PDBsum], [http://www.rcsb.org/pdb/explore.do?structureId=1krj RCSB]</span>
| + | == Function == |
| - | }}
| + | [https://www.uniprot.org/uniprot/CCPR_YEAST CCPR_YEAST] Destroys radicals which are normally produced within the cells and which are toxic to biological systems. |
| | + | == Evolutionary Conservation == |
| | + | [[Image:Consurf_key_small.gif|200px|right]] |
| | + | Check<jmol> |
| | + | <jmolCheckbox> |
| | + | <scriptWhenChecked>; select protein; define ~consurf_to_do selected; consurf_initial_scene = true; script "/wiki/ConSurf/kr/1krj_consurf.spt"</scriptWhenChecked> |
| | + | <scriptWhenUnchecked>script /wiki/extensions/Proteopedia/spt/initialview01.spt</scriptWhenUnchecked> |
| | + | <text>to colour the structure by Evolutionary Conservation</text> |
| | + | </jmolCheckbox> |
| | + | </jmol>, as determined by [http://consurfdb.tau.ac.il/ ConSurfDB]. You may read the [[Conservation%2C_Evolutionary|explanation]] of the method and the full data available from [http://bental.tau.ac.il/new_ConSurfDB/main_output.php?pdb_ID=1krj ConSurf]. |
| | + | <div style="clear:both"></div> |
| | | | |
| - | '''Engineering Calcium-binding site into Cytochrome c Peroxidase (CcP)'''
| + | ==See Also== |
| - | | + | *[[Cytochrome c peroxidase 3D structures|Cytochrome c peroxidase 3D structures]] |
| - | | + | __TOC__ |
| - | ==Overview== | + | </StructureSection> |
| - | 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.
| + | [[Category: Large Structures]] |
| - | | + | |
| - | ==About this Structure==
| + | |
| - | 1KRJ is a [[Single protein]] structure of sequence from [http://en.wikipedia.org/wiki/Saccharomyces_cerevisiae Saccharomyces cerevisiae]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1KRJ 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:[http://www.ncbi.nlm.nih.gov/pubmed/10608846 10608846]
| + | |
| - | [[Category: Cytochrome-c peroxidase]] | + | |
| | [[Category: Saccharomyces cerevisiae]] | | [[Category: Saccharomyces cerevisiae]] |
| - | [[Category: Single protein]]
| + | [[Category: Bhaskar B]] |
| - | [[Category: Bhaskar, B.]] | + | [[Category: Bonagura CA]] |
| - | [[Category: Bonagura, C A.]] | + | [[Category: Poulos TL]] |
| - | [[Category: Poulos, T L.]] | + | [[Category: Sundaramoorthy M]] |
| - | [[Category: Sundaramoorthy, M.]] | + | |
| - | [[Category: bidentate bond]]
| + | |
| - | [[Category: calcium selectivity]]
| + | |
| - | [[Category: cation-binding loop]]
| + | |
| - | [[Category: lignin peroxidase]]
| + | |
| - | [[Category: open/closed conformer]]
| + | |
| - | [[Category: pentagonal bipyrimidal geometry]]
| + | |
| - | [[Category: trp191 cationic-radical]]
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| - | | + | |
| - | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Sun Mar 30 21:52:33 2008''
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