ИСТИНА

Introduction: The possibility of engineering near-infrared fluorescent proteins from bacterial phytochrome photoreceptors (BphPs) has led to substantial interest in this family of proteins. A natural chromophore of BphPs is a heme-derived tetrapyrrole compound biliverdin IXa (BV), which is abundant in mammalian cells. Free BV is non-fluorescent but as a chromophore of BphPs it absorbs and fluoresces in a near-infrared “optical window” (650-900 nm) where mammalian tissues is the most transparent. Thus fluorescent probes developed on the basis of BphPs give the opportunity of non-invasive bio-imaging of deep tissues and whole organs in living animals. Purpose: This work is focused on the study of spectral properties, stability and interaction with the chromophore of the near-infrared iRFP, engineered from bacterial phytochrome RpBphP2. Materials and Methods: We used spectroscopic techniques: absorbance, fluorescence, circular dichroism. Results: The iRFP has characteristic near-infrared absorbance and fluorescence peaked at 690 and 715 nm. The denaturation of iRFP and its apoform (BV-free form) induced by guanidine hydrochloride imply that the chromophore significantly stabilizes iRFP and makes the denaturation transition more cooperative. Moreover in contract to apoform the denaturation of iRFP is irreversible and protein renaturation is complicated by the aggregation of protein molecules. Apoprotein correctly binds BV as proved by recovery of near-infrared absorption, fluorescence and CD. Experiments on iRFP mutant with a substitution of Cys15 by Ser which binds the chromophore non-covalently allowed us determining the binding constant by equilibrium microdialysis. Conclusions: Our data showed that near infrared chromophore of iRFP donates to the protein stability but prevents correct protein refolding.