ИСТИНА

Bacterial phytochromes (BphPs) have attracted attention recently as they can be used to construct novel fluorescent probes for bioimaging of living cells and tissues for highly versatile biological applications and medical diagnostics. An optimal fluorescent probe for imaging in tissue should have both excitation and emission spectra in a near-infrared “optical window” (NIRW: ~650-900 nm) where mammalian tissues are the most transparent to light. Recently such fluorescent probes have been developed on the basis of BphPs, including iRFP, IFP1.4. In these probes biliverdin IXα (BV), which is a natural ligand of BphPs, is used as a chromophore. As BV is a component of endogenous mammalian heme metabolism, no exogenous cofactor is needed. Despite the importance of BphPs, little is known on photochemical processes underlying their near-infrared fluorescence and the amino acids stabilizing different chromophore states. The folding of BphPs containing the rare knot structure between domains involved in chromophore binding has not been investigated also. In this work we studied the guanidine hydrochloride induced denaturation-renaturation of iRFP in apo and holo-form using spectroscopic techniques (absorbance, fluorescence, circular dichroism). It was shown that the denaturation of iRFP in apo-form (without BV) is fully reversible as indicated by the recovery of all recorded parameters. The chromophore binding increases the protein stability and denaturation cooperativity but makes the denaturation of iRFP in holo-form irreversible as its renaturation is complicated by aggregation of partially folded protein molecules.