ИСТИНА

Current interest to biliverdin (BV) is caused by its use in the new near-infrared fluorescent probes developed from bacterial phytochrome photoreceptors (BphPs), in which BV is a natural chromophore. Being a heme derivative, BV is abundant in mammalian cells. BV absorbs and fluoresces in a near-infrared “optical window” (650-900 nm) where mammalian tissues are the most transparent. The fluorescent probes developed on the basis of BphPs provide an opportunity for non-invasive imaging of deep tissues and whole organs in living animals [1, 2, 3]. Naturally, BV binding occurs in the chromophore-binding part of BphPs, which consists of so called PAS and GAF domains. The PAS domain in BphPs contains at an N-terminal patch a conserved cysteine residue, which covalently attaches the BV, while the chromophore itself fits into the cleft in the GAF domain. The N-terminal region of 35 residues upstream of the PAS domain and the loop contributed by the GAF domain (residues 225-257) create a figure-of-eight knot structure, which bridges the PAS and GAF domains [4], so that BV pierces through this structure. At the same time it was shown that apoform of iRFP has native structure. Apoprotein correctly binds BV as proved by recovery of near-infrared absorption, fluorescence and CD. Experiments on iRFP mutant with a substitution of Cys15 with Ser allowed us determining the BV binding constant by equilibrium microdialysis. 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 contrast to the apoform, the denaturation of iRFP with bound BV is irreversible, and its renaturation is complicated by the aggregation of protein molecules. [5]. To clear the spectral properties of BV incorporated in proteins we examined its microenvironment in iRFP and studied spectral properties of the free chromophore in solutions with different polarity, viscosity, dielectric constant and pH.