3mno: Difference between revisions
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[[Image: | ==Crystal structure of the agonist form of mouse glucocorticoid receptor stabilized by (A611V, F608S) mutations at 1.55A== | ||
<StructureSection load='3mno' size='340' side='right' caption='[[3mno]], [[Resolution|resolution]] 1.55Å' scene=''> | |||
== Structural highlights == | |||
<table><tr><td colspan='2'>[[3mno]] is a 2 chain structure with sequence from [http://en.wikipedia.org/wiki/Mus_musculus Mus musculus]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=3MNO OCA]. For a <b>guided tour on the structure components</b> use [http://oca.weizmann.ac.il/oca-docs/fgij/fg.htm?mol=3MNO FirstGlance]. <br> | |||
</td></tr><tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat"><scene name='pdbligand=DEX:DEXAMETHASONE'>DEX</scene>, <scene name='pdbligand=GOL:GLYCEROL'>GOL</scene>, <scene name='pdbligand=SCN:THIOCYANATE+ION'>SCN</scene></td></tr> | |||
<tr id='related'><td class="sblockLbl"><b>[[Related_structure|Related:]]</b></td><td class="sblockDat">[[3mne|3mne]], [[3mnp|3mnp]]</td></tr> | |||
<tr id='gene'><td class="sblockLbl"><b>[[Gene|Gene:]]</b></td><td class="sblockDat">Nr3c1, Grl, Grl1 ([http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&srchmode=5&id=10090 Mus musculus])</td></tr> | |||
<tr id='resources'><td class="sblockLbl"><b>Resources:</b></td><td class="sblockDat"><span class='plainlinks'>[http://oca.weizmann.ac.il/oca-docs/fgij/fg.htm?mol=3mno FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=3mno OCA], [http://www.rcsb.org/pdb/explore.do?structureId=3mno RCSB], [http://www.ebi.ac.uk/pdbsum/3mno PDBsum]</span></td></tr> | |||
</table> | |||
== 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/mn/3mno_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/chain_selection.php?pdb_ID=2ata ConSurf]. | |||
<div style="clear:both"></div> | |||
<div style="background-color:#fffaf0;"> | |||
== Publication Abstract from PubMed == | |||
The human glucocorticoid receptor ligand-binding domain (hGR-LBD) is an important drug target for the treatment of various diseases. However, the low intrinsic stability and solubility of hGR-LBD have rendered its purification and biophysical characterization difficult. In order to overcome these problems, we have stabilized hGR-LBD by a combination of random mutagenesis and high-throughput screening using fluorescence-activated cell sorting (FACS) with enhanced green fluorescent protein (eGFP) as folding reporter. Two plasmid-encoded gene libraries of hGR-LBD fused to the egfp gene were expressed in Escherichia coli, followed by eight rounds of FACS screening, in each of which 10(8) cells were analyzed. The hgr-lbd mutants isolated by this approach contained numerous amino acid exchanges, and four beneficial ones (A605V, V702A, E705G, and M752T) were followed up in detail. Their characterization showed that the fluorescence of hGR-LBD-eGFP fusions is correlated linearly with the stability and solubility of hGR-LBD in the absence of eGFP. When combined, the four exchanges increased the thermal stability of hGR-LBD by more than 8 degrees C and enhanced its purification yield after expression in E. coli by about 26-fold. The introduction of three beneficial exchanges into the homologous ligand-binding domain of mouse enabled its X-ray structure determination at high resolution, which showed how the exchanges stabilize the protein and revealed atomic details that will guide future drug design. Our results demonstrate that large eGFP fusion libraries can be screened by FACS with extreme sensitivity and efficiency, yielding stabilized eukaryotic proteins suitable for biophysical characterization and structure determination. | |||
Enhancing the stability and solubility of the glucocorticoid receptor ligand-binding domain by high-throughput library screening.,Seitz T, Thoma R, Schoch GA, Stihle M, Benz J, D'Arcy B, Wiget A, Ruf A, Hennig M, Sterner R J Mol Biol. 2010 Nov 5;403(4):562-77. Epub 2010 Sep 17. PMID:20850457<ref>PMID:20850457</ref> | |||
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.<br> | |||
</div> | |||
==See Also== | ==See Also== | ||
*[[Glucocorticoid receptor|Glucocorticoid receptor]] | *[[Glucocorticoid receptor|Glucocorticoid receptor]] | ||
*[[Hormone|Hormone]] | *[[Hormone|Hormone]] | ||
== References == | |||
== | <references/> | ||
< | __TOC__ | ||
</StructureSection> | |||
[[Category: Mus musculus]] | [[Category: Mus musculus]] | ||
[[Category: Arcy, B D | [[Category: Arcy, B D]] | ||
[[Category: Banner, D | [[Category: Banner, D]] | ||
[[Category: Benz, J | [[Category: Benz, J]] | ||
[[Category: Hennig, M | [[Category: Hennig, M]] | ||
[[Category: Ruf, A | [[Category: Ruf, A]] | ||
[[Category: Schoch, G A | [[Category: Schoch, G A]] | ||
[[Category: Seitz, T | [[Category: Seitz, T]] | ||
[[Category: Sterner, R | [[Category: Sterner, R]] | ||
[[Category: Stihle, M | [[Category: Stihle, M]] | ||
[[Category: Thoma, R | [[Category: Thoma, R]] | ||
[[Category: Agonist]] | [[Category: Agonist]] | ||
[[Category: Co-activator]] | [[Category: Co-activator]] | ||
Revision as of 11:32, 9 December 2014
Crystal structure of the agonist form of mouse glucocorticoid receptor stabilized by (A611V, F608S) mutations at 1.55ACrystal structure of the agonist form of mouse glucocorticoid receptor stabilized by (A611V, F608S) mutations at 1.55A
Structural highlights
Evolutionary Conservation Check, as determined by ConSurfDB. You may read the explanation of the method and the full data available from ConSurf. Publication Abstract from PubMedThe human glucocorticoid receptor ligand-binding domain (hGR-LBD) is an important drug target for the treatment of various diseases. However, the low intrinsic stability and solubility of hGR-LBD have rendered its purification and biophysical characterization difficult. In order to overcome these problems, we have stabilized hGR-LBD by a combination of random mutagenesis and high-throughput screening using fluorescence-activated cell sorting (FACS) with enhanced green fluorescent protein (eGFP) as folding reporter. Two plasmid-encoded gene libraries of hGR-LBD fused to the egfp gene were expressed in Escherichia coli, followed by eight rounds of FACS screening, in each of which 10(8) cells were analyzed. The hgr-lbd mutants isolated by this approach contained numerous amino acid exchanges, and four beneficial ones (A605V, V702A, E705G, and M752T) were followed up in detail. Their characterization showed that the fluorescence of hGR-LBD-eGFP fusions is correlated linearly with the stability and solubility of hGR-LBD in the absence of eGFP. When combined, the four exchanges increased the thermal stability of hGR-LBD by more than 8 degrees C and enhanced its purification yield after expression in E. coli by about 26-fold. The introduction of three beneficial exchanges into the homologous ligand-binding domain of mouse enabled its X-ray structure determination at high resolution, which showed how the exchanges stabilize the protein and revealed atomic details that will guide future drug design. Our results demonstrate that large eGFP fusion libraries can be screened by FACS with extreme sensitivity and efficiency, yielding stabilized eukaryotic proteins suitable for biophysical characterization and structure determination. Enhancing the stability and solubility of the glucocorticoid receptor ligand-binding domain by high-throughput library screening.,Seitz T, Thoma R, Schoch GA, Stihle M, Benz J, D'Arcy B, Wiget A, Ruf A, Hennig M, Sterner R J Mol Biol. 2010 Nov 5;403(4):562-77. Epub 2010 Sep 17. PMID:20850457[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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