3ED8

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

3ED8 の概要

• エントリーDOI: 10.2210/pdb3ed8/pdb
• 分子名称: yellow fluorescence protein (2 entities in total)
• 機能のキーワード: superfolder, bimolecular fluorescence complementation, luminescent protein
• 由来する生物種: Aequorea victoria
• タンパク質・核酸の鎖数: 5
• 化学式量合計: 146696.35

構造登録者

Ottmann, C.,Weyand, M. (登録日: 2008-09-02, 公開日: 2009-01-20, 最終更新日: 2024-11-06)

主引用文献

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

実験手法

X-RAY DIFFRACTION (2.7 Å)