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A disulphide bond-mediated hetero-dimer of a hemoprotein and a fluorescent protein exhibiting efficient energy transfer† † Electronic supplementary information (ESI) available. See https://doi.org/10.1039/d2ra05249k

ABSTRACT: Artificial protein hetero-dimerization is one of the promising strategies to construct protein-based chemical tools. In this work, cytochrome b562, 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. 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.

SUBMITTER: Soon J

PROVIDER: S-EPMC9535469 | biostudies-literature | 2022 Oct

REPOSITORIES: biostudies-literature

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Project description:Photophysical data and orbital energy levels (from electrochemistry) were compared for molecules with the same BODIPY acceptor part (red) and perpendicularly oriented xanthene or BODIPY donor fragments (green). Transfer of energy, hence the photophysical properties of the cassettes, including the pH dependent fluorescence in the xanthene-containing molecules, correlates with the relative energies of the frontier orbitals in these systems. Intracellular sensing of protons is often achieved via sensors that switch off completely at certain pH values, but probes of this type are not easy to locate inside cells in their "off-state". A communication from these laboratories (J. Am. Chem. Soc., 2009, 131, 1642-3) described how the energy transfer cassette 1 could be used for intracellular imaging of pH. This probe is fluorescent whatever the pH, but its exact photophysical properties are governed by the protonation states of the xanthene donors. This work was undertaken to further investigate correlations between structure, photophysical properties, and pH for energy transfer cassettes. To achieve this, three other cassettes 2-4 were prepared: another one containing pH-sensitive xanthene donors (2) and two "control cassettes" that each have two BODIPY-based donors (3 and 4). Both the cassettes 1 and 2 with xanthene-based donors fluoresce red under slightly acidic conditions (pH < ∼6) and green when the medium is more basic (>∼7), whereas the corresponding cassettes with BODIPY donors give almost complete energy transfer regardless of pH. The cassettes that have BODIPY donors, by contrast, show no significant fluorescence from the donor parts, but the overall quantum yields of the cassettes when excited at the donor (observation of acceptor fluorescence) are high (ca. 0.6 and 0.9). Electrochemical measurements were performed to elucidate orbital energy level differences between the pH-fluorescence profiles of cassettes with xanthene donors, relative to the two with BODIPY donors. These studies confirm energy transfer in the cassettes is dramatically altered by analytes that perturb relative orbital levels. Energy transfer cassettes with distinct fluorescent donor and acceptor units provide a new, and potentially useful, approach to sensors for biomedical applications.

Dataset Information

A disulphide bond-mediated hetero-dimer of a hemoprotein and a fluorescent protein exhibiting efficient energy transfer† † Electronic supplementary information (ESI) available. See https://doi.org/10.1039/d2ra05249k

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