1dfa: Difference between revisions
New page: left|200px<br /><applet load="1dfa" size="450" color="white" frame="true" align="right" spinBox="true" caption="1dfa, resolution 2.0Å" /> '''CRYSTAL STRUCTURE OF ... |
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[[Image:1dfa.gif|left|200px]]<br /><applet load="1dfa" size=" | [[Image:1dfa.gif|left|200px]]<br /><applet load="1dfa" size="350" color="white" frame="true" align="right" spinBox="true" | ||
caption="1dfa, resolution 2.0Å" /> | caption="1dfa, resolution 2.0Å" /> | ||
'''CRYSTAL STRUCTURE OF PI-SCEI IN C2 SPACE GROUP'''<br /> | '''CRYSTAL STRUCTURE OF PI-SCEI IN C2 SPACE GROUP'''<br /> | ||
==Overview== | ==Overview== | ||
The PI-SceI protein is an intein-encoded homing endonuclease that | The PI-SceI protein is an intein-encoded homing endonuclease that initiates the mobility of its gene by making a double strand break at a single site in the yeast genome. The PI-SceI protein splicing and endonucleolytic active sites are separately located in each of two domains in the PI-SceI structure. To determine the spatial relationship between bases in the PI-SceI recognition sequence and selected PI-SceI amino acids, the PI-SceI-DNA complex was probed by photocross-linking and affinity cleavage methods. Unique solvent-accessible cysteine residues were introduced into the two PI-SceI domains at positions 91, 97, 170, 230, 376, and 378, and the mutant proteins were modified with either 4-azidophenacyl bromide or iron (S)-1-(p-bromoacetamidobenzyl)-ethylenediaminetetraacetate (FeBABE). The phenyl azide-coupled proteins cross-linked to the PI-SceI target sequence, and the FeBABE-modified proteins cleaved the DNA proximal to the derivatized amino acid. The results suggest that an extended beta-hairpin loop in the endonuclease domain that contains residues 376 and 378 contacts the major groove near the PI-SceI cleavage site. Conversely, residues 91, 97, and 170 in the protein splicing domain are in close proximity to a distant region of the substrate. To interpret our results, we used a new PI-SceI structure that is ordered in regions of the protein that bind DNA. The data strongly support a model of the PI-SceI-DNA complex derived from this structure. | ||
==About this Structure== | ==About this Structure== | ||
1DFA is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Saccharomyces_cerevisiae Saccharomyces cerevisiae]. Active as [http://en.wikipedia.org/wiki/H(+)-transporting_two-sector_ATPase H(+)-transporting two-sector ATPase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.6.3.14 3.6.3.14] Full crystallographic information is available from [http:// | 1DFA is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Saccharomyces_cerevisiae Saccharomyces cerevisiae]. Active as [http://en.wikipedia.org/wiki/H(+)-transporting_two-sector_ATPase H(+)-transporting two-sector ATPase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.6.3.14 3.6.3.14] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1DFA OCA]. | ||
==Reference== | ==Reference== | ||
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[[Category: Crist, M.]] | [[Category: Crist, M.]] | ||
[[Category: Duan, X.]] | [[Category: Duan, X.]] | ||
[[Category: Gimble, F | [[Category: Gimble, F S.]] | ||
[[Category: Hu, D.]] | [[Category: Hu, D.]] | ||
[[Category: Quiocho, F | [[Category: Quiocho, F A.]] | ||
[[Category: homing endonuclease]] | [[Category: homing endonuclease]] | ||
[[Category: hydrolase]] | [[Category: hydrolase]] | ||
[[Category: intein]] | [[Category: intein]] | ||
''Page seeded by [http:// | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 12:16:05 2008'' | ||
Revision as of 13:16, 21 February 2008
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CRYSTAL STRUCTURE OF PI-SCEI IN C2 SPACE GROUP
OverviewOverview
The PI-SceI protein is an intein-encoded homing endonuclease that initiates the mobility of its gene by making a double strand break at a single site in the yeast genome. The PI-SceI protein splicing and endonucleolytic active sites are separately located in each of two domains in the PI-SceI structure. To determine the spatial relationship between bases in the PI-SceI recognition sequence and selected PI-SceI amino acids, the PI-SceI-DNA complex was probed by photocross-linking and affinity cleavage methods. Unique solvent-accessible cysteine residues were introduced into the two PI-SceI domains at positions 91, 97, 170, 230, 376, and 378, and the mutant proteins were modified with either 4-azidophenacyl bromide or iron (S)-1-(p-bromoacetamidobenzyl)-ethylenediaminetetraacetate (FeBABE). The phenyl azide-coupled proteins cross-linked to the PI-SceI target sequence, and the FeBABE-modified proteins cleaved the DNA proximal to the derivatized amino acid. The results suggest that an extended beta-hairpin loop in the endonuclease domain that contains residues 376 and 378 contacts the major groove near the PI-SceI cleavage site. Conversely, residues 91, 97, and 170 in the protein splicing domain are in close proximity to a distant region of the substrate. To interpret our results, we used a new PI-SceI structure that is ordered in regions of the protein that bind DNA. The data strongly support a model of the PI-SceI-DNA complex derived from this structure.
About this StructureAbout this Structure
1DFA is a Single protein structure of sequence from Saccharomyces cerevisiae. Active as H(+)-transporting two-sector ATPase, with EC number 3.6.3.14 Full crystallographic information is available from OCA.
ReferenceReference
Probing the structure of the PI-SceI-DNA complex by affinity cleavage and affinity photocross-linking., Hu D, Crist M, Duan X, Quiocho FA, Gimble FS, J Biol Chem. 2000 Jan 28;275(4):2705-12. PMID:10644733
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