1pae
From Proteopedia
(New page: 200px<br /><applet load="1pae" size="450" color="white" frame="true" align="right" spinBox="true" caption="1pae, resolution 2.70Å" /> '''nucleoside diphospha...) |
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| - | [[Image:1pae.gif|left|200px]]<br /><applet load="1pae" size=" | + | [[Image:1pae.gif|left|200px]]<br /><applet load="1pae" size="350" color="white" frame="true" align="right" spinBox="true" |
caption="1pae, resolution 2.70Å" /> | caption="1pae, resolution 2.70Å" /> | ||
'''nucleoside diphosphate kinase'''<br /> | '''nucleoside diphosphate kinase'''<br /> | ||
==Overview== | ==Overview== | ||
| - | A protocol for the quantitative incorporation of both selenomethionine and | + | A protocol for the quantitative incorporation of both selenomethionine and selenocysteine into recombinant proteins overexpressed in Escherichia coli is described. This methodology is based on the use of a suitable cysteine auxotrophic strain and a minimal medium supplemented with selenium-labeled methionine and cysteine. The proteins chosen for these studies are the cathelin-like motif of protegrin-3 and a nucleoside-diphosphate kinase. Analysis of the purified proteins by electrospray mass spectrometry and X-ray crystallography revealed that both cysteine and methionine residues were isomorphously replaced by selenocysteine and selenomethionine. Moreover, selenocysteines allowed the formation of unstrained and stable diselenide bridges in place of the canonical disulfide bonds. In addition, we showed that NDP kinase contains a selenocysteine adduct on Cys122. This novel selenium double-labeling method is proposed as a general approach to increase the efficiency of the MAD technique used for phase determination in protein crystallography. |
==About this Structure== | ==About this Structure== | ||
| - | 1PAE is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Dictyostelium_discoideum Dictyostelium discoideum] with SE as [http://en.wikipedia.org/wiki/ligand ligand]. Active as [http://en.wikipedia.org/wiki/Nucleoside-diphosphate_kinase Nucleoside-diphosphate kinase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=2.7.4.6 2.7.4.6] Full crystallographic information is available from [http:// | + | 1PAE is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Dictyostelium_discoideum Dictyostelium discoideum] with <scene name='pdbligand=SE:'>SE</scene> as [http://en.wikipedia.org/wiki/ligand ligand]. Active as [http://en.wikipedia.org/wiki/Nucleoside-diphosphate_kinase Nucleoside-diphosphate kinase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=2.7.4.6 2.7.4.6] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1PAE OCA]. |
==Reference== | ==Reference== | ||
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[[Category: Dumas, C.]] | [[Category: Dumas, C.]] | ||
[[Category: Hoh, F.]] | [[Category: Hoh, F.]] | ||
| - | [[Category: Sanchez, J | + | [[Category: Sanchez, J F.]] |
| - | [[Category: Strub, J | + | [[Category: Strub, J M.]] |
| - | [[Category: Strub, M | + | [[Category: Strub, M P.]] |
[[Category: SE]] | [[Category: SE]] | ||
[[Category: selenocysteine selenomethionine]] | [[Category: selenocysteine selenomethionine]] | ||
| - | ''Page seeded by [http:// | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 14:26:48 2008'' |
Revision as of 12:27, 21 February 2008
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nucleoside diphosphate kinase
Overview
A protocol for the quantitative incorporation of both selenomethionine and selenocysteine into recombinant proteins overexpressed in Escherichia coli is described. This methodology is based on the use of a suitable cysteine auxotrophic strain and a minimal medium supplemented with selenium-labeled methionine and cysteine. The proteins chosen for these studies are the cathelin-like motif of protegrin-3 and a nucleoside-diphosphate kinase. Analysis of the purified proteins by electrospray mass spectrometry and X-ray crystallography revealed that both cysteine and methionine residues were isomorphously replaced by selenocysteine and selenomethionine. Moreover, selenocysteines allowed the formation of unstrained and stable diselenide bridges in place of the canonical disulfide bonds. In addition, we showed that NDP kinase contains a selenocysteine adduct on Cys122. This novel selenium double-labeling method is proposed as a general approach to increase the efficiency of the MAD technique used for phase determination in protein crystallography.
About this Structure
1PAE is a Single protein structure of sequence from Dictyostelium discoideum with as ligand. Active as Nucleoside-diphosphate kinase, with EC number 2.7.4.6 Full crystallographic information is available from OCA.
Reference
Selenomethionine and selenocysteine double labeling strategy for crystallographic phasing., Strub MP, Hoh F, Sanchez JF, Strub JM, Bock A, Aumelas A, Dumas C, Structure. 2003 Nov;11(11):1359-67. PMID:14604526
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