Abstract
Fluorescent proteins (FPs) engineered from bacterial phytochromes attract attention as probes for in vivo imaging due to their near-infrared (NIR) spectra and use of available in mammalian cells biliverdin (BV) as chromophore. We studied spectral properties of the iRFP670, iRFP682 and iRFP713 proteins and their mutants having Cys residues able to bind BV either in both PAS (Cys15) and GAF (Cys256) domains, in one of these domains, or without these Cys residues. We show that the absorption and fluorescence spectra and the chromophore binding depend on the location of the Cys residues. Compared with NIR FPs in which BV covalently binds to Cys15 or those that incorporate BV noncovalently, the proteins with BV covalently bound to Cys256 have blue-shifted spectra and higher quantum yield. In dimeric NIR FPs without Cys15, the covalent binding of BV to Сys256 in one monomer allosterically inhibits the covalent binding of BV to the other monomer, whereas the presence of Cys15 allosterically promotes BV binding to Cys256 in both monomers. The NIR FPs with both Cys residues have the narrowest blue-shifted spectra and the highest quantum yield. Our analysis resulted in the iRFP713/Val256Cys protein with the highest brightness in mammalian cells among available NIR FPs.
Highlights
The dependences of the chromophore absorbance, fluorescence and ellipticity at 222 nm on the GdnHCl concentration for the iRFP670, iRFP682 and iRFP713 variants were recorded at 23 °C after protein incubation in a solution of an appropriate denaturant concentration at 23 °С for 24 h
To construct mammalian expression plasmids, piRFP670, piRFP682, piRFP670/C15S, piRFP670/C256S, piRFP670/C15S/C256S, piRFP682/C15S, piRFP682/C256S, piRFP682/C15S/ C256S, piRFP713 and piRFP713/V256C the respective genes were PCR-amplified as AgeI-NotI fragments and swapped with a gene encoding EGFP in the pEGFP-N1 plasmid (Clontech)
HeLa cells were grown in DMEM medium supplemented with 10% FBS, 0.5% penicillin-streptomycin and 2 mM glutamine (Life Technologies/Invitrogen)
Summary
The dependences of the chromophore absorbance, fluorescence and ellipticity at 222 nm on the GdnHCl concentration for the iRFP670, iRFP682 and iRFP713 variants were recorded at 23 °C after protein incubation in a solution of an appropriate denaturant concentration at 23 °С for 24 h. The recorded fluorescence intensity was corrected for the total optical density of the solution (DΣ).
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