5ma4 | pdb_00005ma4

GFP-binding DARPin fusion gc_K7

spinqualitylabelsall models 5ma4, resolution 1.40Å Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D Structural highlights 5ma4 is a 2 chain structure with sequence from Aeqvi and Synthetic construct sequences. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance. NonStd Res: CRO Gene: GFP (AEQVI) 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 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 From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.

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