5ma3: Difference between revisions
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<StructureSection load='5ma3' size='340' side='right' caption='[[5ma3]], [[Resolution|resolution]] 1.70Å' scene=''> | <StructureSection load='5ma3' size='340' side='right' caption='[[5ma3]], [[Resolution|resolution]] 1.70Å' scene=''> | ||
== Structural highlights == | == Structural highlights == | ||
<table><tr><td colspan='2'>[[5ma3]] is a 2 chain structure. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=5MA3 OCA]. For a <b>guided tour on the structure components</b> use [http://oca.weizmann.ac.il/oca-docs/fgij/fg.htm?mol=5MA3 FirstGlance]. <br> | <table><tr><td colspan='2'>[[5ma3]] is a 2 chain structure with sequence from [http://en.wikipedia.org/wiki/Aeqvi Aeqvi] and [http://en.wikipedia.org/wiki/Synthetic_construct_sequences Synthetic construct sequences]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=5MA3 OCA]. For a <b>guided tour on the structure components</b> use [http://oca.weizmann.ac.il/oca-docs/fgij/fg.htm?mol=5MA3 FirstGlance]. <br> | ||
</td></tr><tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat"><scene name='pdbligand=EDO:1,2-ETHANEDIOL'>EDO</scene></td></tr> | </td></tr><tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat"><scene name='pdbligand=EDO:1,2-ETHANEDIOL'>EDO</scene></td></tr> | ||
<tr id='NonStdRes'><td class="sblockLbl"><b>[[Non-Standard_Residue|NonStd Res:]]</b></td><td class="sblockDat"><scene name='pdbligand=CRO:{2-[(1R,2R)-1-AMINO-2-HYDROXYPROPYL]-4-(4-HYDROXYBENZYLIDENE)-5-OXO-4,5-DIHYDRO-1H-IMIDAZOL-1-YL}ACETIC+ACID'>CRO</scene></td></tr> | <tr id='NonStdRes'><td class="sblockLbl"><b>[[Non-Standard_Residue|NonStd Res:]]</b></td><td class="sblockDat"><scene name='pdbligand=CRO:{2-[(1R,2R)-1-AMINO-2-HYDROXYPROPYL]-4-(4-HYDROXYBENZYLIDENE)-5-OXO-4,5-DIHYDRO-1H-IMIDAZOL-1-YL}ACETIC+ACID'>CRO</scene></td></tr> | ||
<tr id='gene'><td class="sblockLbl"><b>[[Gene|Gene:]]</b></td><td class="sblockDat">GFP ([http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&srchmode=5&id=6100 AEQVI])</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=5ma3 FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=5ma3 OCA], [http://pdbe.org/5ma3 PDBe], [http://www.rcsb.org/pdb/explore.do?structureId=5ma3 RCSB], [http://www.ebi.ac.uk/pdbsum/5ma3 PDBsum], [http://prosat.h-its.org/prosat/prosatexe?pdbcode=5ma3 ProSAT]</span></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=5ma3 FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=5ma3 OCA], [http://pdbe.org/5ma3 PDBe], [http://www.rcsb.org/pdb/explore.do?structureId=5ma3 RCSB], [http://www.ebi.ac.uk/pdbsum/5ma3 PDBsum], [http://prosat.h-its.org/prosat/prosatexe?pdbcode=5ma3 ProSAT]</span></td></tr> | ||
</table> | </table> | ||
== Function == | == Function == | ||
[[http://www.uniprot.org/uniprot/GFP_AEQVI GFP_AEQVI]] Energy-transfer acceptor. Its role is to transduce the blue chemiluminescence of the protein aequorin into green fluorescent light by energy transfer. Fluoresces in vivo upon receiving energy from the Ca(2+)-activated photoprotein aequorin. | [[http://www.uniprot.org/uniprot/GFP_AEQVI GFP_AEQVI]] Energy-transfer acceptor. Its role is to transduce the blue chemiluminescence of the protein aequorin into green fluorescent light by energy transfer. Fluoresces in vivo upon receiving energy from the Ca(2+)-activated photoprotein aequorin. | ||
<div style="background-color:#fffaf0;"> | |||
== Publication Abstract from PubMed == | |||
Green fluorescent protein (GFP) fusions are pervasively used to study structures and processes. Specific GFP-binders are thus of great utility for detection, immobilization or manipulation of GFP-fused molecules. We determined structures of two designed ankyrin repeat proteins (DARPins), complexed with GFP, which revealed different but overlapping epitopes. Here we show a structure-guided design strategy that, by truncation and computational reengineering, led to a stable construct where both can bind simultaneously: by linkage of the two binders, fusion constructs were obtained that "wrap around" GFP, have very high affinities of about 10-30 pM, and extremely slow off-rates. They can be natively produced in E. coli in very large amounts, and show excellent biophysical properties. Their very high stability and affinity, facile site-directed functionalization at introduced unique lysines or cysteines facilitate many applications. As examples, we present them as tight yet reversible immobilization reagents for surface plasmon resonance, as fluorescently labelled monomeric detection reagents in flow cytometry, as pull-down ligands to selectively enrich GFP fusion proteins from cell extracts, and as affinity column ligands for inexpensive large-scale protein purification. We have thus described a general design strategy to create a "clamp" from two different high-affinity repeat proteins, even if their epitopes overlap. | |||
Design and applications of a clamp for Green Fluorescent Protein with picomolar affinity.,Hansen S, Stuber JC, Ernst P, Koch A, Bojar D, Batyuk A, Pluckthun A Sci Rep. 2017 Nov 24;7(1):16292. doi: 10.1038/s41598-017-15711-z. PMID:29176615<ref>PMID:29176615</ref> | |||
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.<br> | |||
</div> | |||
<div class="pdbe-citations 5ma3" style="background-color:#fffaf0;"></div> | |||
== References == | |||
<references/> | |||
__TOC__ | __TOC__ | ||
</StructureSection> | </StructureSection> | ||
[[Category: Aeqvi]] | |||
[[Category: Synthetic construct sequences]] | |||
[[Category: Batyuk, A]] | [[Category: Batyuk, A]] | ||
[[Category: Bojar, D]] | [[Category: Bojar, D]] | ||
Revision as of 10:40, 6 December 2017
GFP-binding DARPin fusion gc_R11GFP-binding DARPin fusion gc_R11
Structural highlights
Function[GFP_AEQVI] Energy-transfer acceptor. Its role is to transduce the blue chemiluminescence of the protein aequorin into green fluorescent light by energy transfer. Fluoresces in vivo upon receiving energy from the Ca(2+)-activated photoprotein aequorin. Publication Abstract from PubMedGreen fluorescent protein (GFP) fusions are pervasively used to study structures and processes. Specific GFP-binders are thus of great utility for detection, immobilization or manipulation of GFP-fused molecules. We determined structures of two designed ankyrin repeat proteins (DARPins), complexed with GFP, which revealed different but overlapping epitopes. Here we show a structure-guided design strategy that, by truncation and computational reengineering, led to a stable construct where both can bind simultaneously: by linkage of the two binders, fusion constructs were obtained that "wrap around" GFP, have very high affinities of about 10-30 pM, and extremely slow off-rates. They can be natively produced in E. coli in very large amounts, and show excellent biophysical properties. Their very high stability and affinity, facile site-directed functionalization at introduced unique lysines or cysteines facilitate many applications. As examples, we present them as tight yet reversible immobilization reagents for surface plasmon resonance, as fluorescently labelled monomeric detection reagents in flow cytometry, as pull-down ligands to selectively enrich GFP fusion proteins from cell extracts, and as affinity column ligands for inexpensive large-scale protein purification. We have thus described a general design strategy to create a "clamp" from two different high-affinity repeat proteins, even if their epitopes overlap. Design and applications of a clamp for Green Fluorescent Protein with picomolar affinity.,Hansen S, Stuber JC, Ernst P, Koch A, Bojar D, Batyuk A, Pluckthun A Sci Rep. 2017 Nov 24;7(1):16292. doi: 10.1038/s41598-017-15711-z. PMID:29176615[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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