A disulphide bond-mediated hetero-dimer of a hemoprotein and a fluorescent protein exhibiting efficient energy transfer. Issue 44 (6th October 2022)

• Record Type: Journal Article
• Authors: Soon, Julian Wong; Oohora, Koji; Hayashi, Takashi
• Is Part Of: RSC advances. Volume 12:Issue 44(2022)
• Page Start: 28519
• Page End: 28524
• Publication Date: 2022-10-06
• Subjects: Chemistry -- Periodicals; 540.5
• DOI: 10.1039/d2ra05249k
• Languages: English
• ISSNs: 2046-2069

Abstract

Abstract : Hetero-dimerization of a hemoprotein and green fluorescent protein via a thiol–disulphide exchange reaction is achieved. The heterodimer has suitable cross-linking points and displays efficient energy transfer. Abstract : Artificial protein hetero-dimerization is one of the promising strategies to construct protein-based chemical tools. In this work, cytochrome b 562, an electron transfer hemoprotein, and green fluorescent protein (GFP) mutants with cysteine residues added to their surfaces were conjugated via a pyridyl disulphide-based thiol–disulfide exchange reaction. The eight hetero-dimers, which have cysteine residues at different positions to form the disulphide bonds, were obtained and characterized by gel-electrophoresis, mass spectrometry and size exclusion chromatography. The fluorescence properties of the hetero-dimers were evaluated by fluorescence spectroscopy and fluorescence lifetime measurements. Efficient photoinduced energy transfer from the GFP chromophore to the heme cofactor was observed in each of the hetero-dimers. The energy transfer efficiency is strongly dependent on the cross-linking residues, reaching 96%. Furthermore, the estimated Förster distance and the structure-based maximum possible distances of the donor and acceptor suggest that one of the hetero-dimers has a rigid protein–protein structure with favourable properties for energy transfer. The disulphide bond-mediated protein hetero-dimerization is useful for screening functional protein systems towards further developments.