3ed8 | pdb_00003ed8

Application of the superfolder YFP bimolecular fluorescence complementation for studying protein-protein interactions in vitro

spinqualitylabelsall models 3ed8, resolution 2.70Å Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D Structural highlights 3ed8 is a 5 chain structure with sequence from Aequorea victoria. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance. Method: X-ray diffraction, Resolution 2.7Å 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. 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 PubMed Abstract Bimolecular fluorescence complementation (BiFC) using yellow fluorescent protein (YFP) is a widely employed method to study protein-protein interactions in cells. As yet, this technique has not been used in vitro. To evaluate a possible application of BiFC in vitro, we constructed a 'superfolder split YFP' system where 15 mutations enhance expression of the fusion proteins in Escherichia coli and enable a native purification due to improved solubility. Here, we present the crystal structure of 'superfolder YFP', providing the structural basis for the enhanced folding and stability characteristics. Complementation between the two non-fluorescent YFP fragments fused to HRas and Raf1RBD or to 14-3-3 and PMA2-CT52 resulted in the constitution of the functional fluorophore. The in vivo BiFC with these protein interaction pairs was demonstrated in eukaryotic cell lines as well. Here, we present for the first time BiFC in vitro studies with natively purified superfolder YFP fusion proteins and show the potential and drawbacks of this method for analyzing protein-protein interactions. Applicability of superfolder YFP bimolecular fluorescence complementation in vitro.,Ottmann C, Weyand M, Wolf A, Kuhlmann J, Ottmann C Biol Chem. 2009 Jan;390(1):81-90. PMID:19007309 From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See Also Green Fluorescent Protein 3D structures

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