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==Crystal structure of GFPuv complexed with the nanobody LaG16 at 1.67 Angstron resolution== | ==Crystal structure of GFPuv complexed with the nanobody LaG16 at 1.67 Angstron resolution== | ||
<StructureSection load='6lr7' size='340' side='right'caption='[[6lr7]]' scene=''> | <StructureSection load='6lr7' size='340' side='right'caption='[[6lr7]], [[Resolution|resolution]] 1.67Å' scene=''> | ||
== Structural highlights == | == Structural highlights == | ||
<table><tr><td colspan='2'>Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=6LR7 OCA]. For a <b>guided tour on the structure components</b> use [ | <table><tr><td colspan='2'>[[6lr7]] is a 2 chain structure with sequence from [https://en.wikipedia.org/wiki/Aequorea_victoria Aequorea victoria] and [https://en.wikipedia.org/wiki/Camelus_bactrianus Camelus bactrianus]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=6LR7 OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=6LR7 FirstGlance]. <br> | ||
</td></tr><tr id='resources'><td class="sblockLbl"><b>Resources:</b></td><td class="sblockDat"><span class='plainlinks'>[ | </td></tr><tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat" id="ligandDat"><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='resources'><td class="sblockLbl"><b>Resources:</b></td><td class="sblockDat"><span class='plainlinks'>[https://proteopedia.org/fgij/fg.htm?mol=6lr7 FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=6lr7 OCA], [https://pdbe.org/6lr7 PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=6lr7 RCSB], [https://www.ebi.ac.uk/pdbsum/6lr7 PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=6lr7 ProSAT]</span></td></tr> | |||
</table> | </table> | ||
== Function == | |||
[https://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 proteins (GFPs) are widely used in biological research. Although GFP can be visualized easily, its precise manipulation through binding partners is still burdensome because of the limited availability of high-affinity binding partners and related structural information. Here, we report the crystal structure of GFPuv in complex with the anti-GFP nanobody LaG16 at 1.67 A resolution, revealing the details of the binding between GFPuv and LaG16. The LaG16 binding site was on the opposite side of the GFP beta-barrel from the binding site of the GFP-enhancer, another anti-GFP nanobody, indicating that the GFP-enhancer and LaG16 can bind to GFP together. Thus, we further designed 3 linkers of different lengths to fuse LaG16 and GFP-enhancer together, and the GFP binding of the three constructs was further tested by ITC. The construct with the (GGGGS)4 linker had the highest affinity with a KD of 0.5 nM. The GFP-enhancer-(GGGGS)4-LaG16 chimeric nanobody was further covalently linked to NHS-activated agarose and then used in the purification of a GFP-tagged membrane protein, GFP-tagged zebrafish P2X4, resulting in higher yield than purification with the GFP-enhancer nanobody alone. This work provides a proof of concept for the design of ultra-high-affinity binders of target proteins through dimerized nanobody chimaeras, and this strategy may also be applied to link interesting target protein nanobodies without overlapping binding surfaces. | |||
Structure-based engineering of anti-GFP nanobody tandems as ultra-high-affinity reagents for purification.,Zhang Z, Wang Y, Ding Y, Hattori M Sci Rep. 2020 Apr 10;10(1):6239. doi: 10.1038/s41598-020-62606-7. PMID:32277083<ref>PMID:32277083</ref> | |||
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.<br> | |||
</div> | |||
<div class="pdbe-citations 6lr7" style="background-color:#fffaf0;"></div> | |||
==See Also== | |||
*[[Antibody 3D structures|Antibody 3D structures]] | |||
*[[Green Fluorescent Protein 3D structures|Green Fluorescent Protein 3D structures]] | |||
== References == | |||
<references/> | |||
__TOC__ | __TOC__ | ||
</StructureSection> | </StructureSection> | ||
[[Category: Aequorea victoria]] | |||
[[Category: Camelus bactrianus]] | |||
[[Category: Large Structures]] | [[Category: Large Structures]] | ||
[[Category: Ding Y]] | [[Category: Ding Y]] | ||
[[Category: Hattori M]] | [[Category: Hattori M]] | ||
[[Category: Zhang ZY]] | [[Category: Zhang ZY]] | ||
Revision as of 09:35, 7 April 2023
Crystal structure of GFPuv complexed with the nanobody LaG16 at 1.67 Angstron resolutionCrystal structure of GFPuv complexed with the nanobody LaG16 at 1.67 Angstron resolution
Structural highlights
FunctionGFP_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 proteins (GFPs) are widely used in biological research. Although GFP can be visualized easily, its precise manipulation through binding partners is still burdensome because of the limited availability of high-affinity binding partners and related structural information. Here, we report the crystal structure of GFPuv in complex with the anti-GFP nanobody LaG16 at 1.67 A resolution, revealing the details of the binding between GFPuv and LaG16. The LaG16 binding site was on the opposite side of the GFP beta-barrel from the binding site of the GFP-enhancer, another anti-GFP nanobody, indicating that the GFP-enhancer and LaG16 can bind to GFP together. Thus, we further designed 3 linkers of different lengths to fuse LaG16 and GFP-enhancer together, and the GFP binding of the three constructs was further tested by ITC. The construct with the (GGGGS)4 linker had the highest affinity with a KD of 0.5 nM. The GFP-enhancer-(GGGGS)4-LaG16 chimeric nanobody was further covalently linked to NHS-activated agarose and then used in the purification of a GFP-tagged membrane protein, GFP-tagged zebrafish P2X4, resulting in higher yield than purification with the GFP-enhancer nanobody alone. This work provides a proof of concept for the design of ultra-high-affinity binders of target proteins through dimerized nanobody chimaeras, and this strategy may also be applied to link interesting target protein nanobodies without overlapping binding surfaces. Structure-based engineering of anti-GFP nanobody tandems as ultra-high-affinity reagents for purification.,Zhang Z, Wang Y, Ding Y, Hattori M Sci Rep. 2020 Apr 10;10(1):6239. doi: 10.1038/s41598-020-62606-7. PMID:32277083[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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