6lr7

Crystal structure of GFPuv complexed with the nanobody LaG16 at 1.67 Angstron resolution

spinqualitylabelsall models 6lr7, resolution 1.67Å Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D Structural highlights 6lr7 is a 2 chain structure with sequence from Aequorea victoria and Camelus bactrianus. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance. Method: X-ray diffraction, Resolution 1.67Å Ligands: CRO Resources: FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT 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 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 From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See Also Antibody 3D structures Green Fluorescent Protein 3D structures

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