3ED8

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

Summary for 3ED8

• Entry DOI: 10.2210/pdb3ed8/pdb
• Descriptor: yellow fluorescence protein (2 entities in total)
• Functional Keywords: superfolder, bimolecular fluorescence complementation, luminescent protein
• Biological source: Aequorea victoria
• Total number of polymer chains: 5
• Total formula weight: 146696.35

Authors

Ottmann, C.,Weyand, M. (deposition date: 2008-09-02, release date: 2009-01-20, Last modification date: 2024-11-06)

Primary citation

Ottmann, C.,Weyand, M.,Wolf, A.,Kuhlmann, J.,Ottmann, C.
Applicability of superfolder YFP bimolecular fluorescence complementation in vitro.
Biol.Chem., 390:81-90, 2009

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.

PubMed: 19007309
DOI: 10.1515/BC.2009.008

Experimental method

X-RAY DIFFRACTION (2.7 Å)