ИСТИНА

Near-infrared (NIR) fluorescent proteins (FPs) engineered from bacterial phytochromes (BphPs) with the absorbance and fluorescence in the "NIR transparency window" of biological tissues meet the requirements for probes useful for deep-tissue in vivo imaging. Spectral properties of NIR FPs, composed of two PAS and GAF domains, are affected by the place of covalent attachment of their natural biliverdin chromophore (BV) with BV-CysPAS adduct being more red-shifted with respect to BV-CysGAF adducts. The covalent binding of BV and its linkage place influences the stability of NIR FP holoprotein. The covalent binding of the chromophore in dimeric NIR FPs was proposed to depend on inter-monomer and inter-domain interactions. The fraction of non-covalently bound chromophore in dimeric NIR FPs resulted in decreased quantum yield and brightness of the protein. We studied the biochemical, structural and spectral properties of BphP1-FP NIR FP and its mutants in buffer solutions and in the presence of denaturant. BphP1-FP has both reactive Cys residues (CysPAS and CysGAF) while its mutants bears either CysPAS or CysGAF, or no of them. All BphP1-FP variants were found to be monomeric. The elimination of inter-monomer interactions led to the absence of the non-covalently bound BV in the monomeric BphP1-FP variants. It confirmed the inter-monomeric allosteric influence on the BV interaction with the protein monomers in dimeric NIR FPs. The unfolding of monomeric BphP1-FP variant in apoform was irreversible in contrast to that of dimeric NIR FPs indicating that inter-molecular contacts contribute to folding of NIR FPs. The increased stability demonstrated by BphP1-FP is likely connected with the inter-domain cross-linking of PAS and GAF domains to BV chromophore. We suppose that similar structural changes in dimeric NIR FPs remove inhibition of covalent binding of the BV and increase their stability.