ИСТИНА

The near-infrared fluorescent probes engineered from bacterial phytochrome photoreceptors (BphPs) make an advance in non-invasive imaging of biological processes in vivo. These proteins are able to develop the near-infrared fluorescence binding and utilizing as a chromophore the biliverdin IXa (BV) available in the mammalian cells. BphPs and their derivative probes contain a unique figure-of-eight knot between domains involved in chromophore binding. The BphPs knot is supposed to be important for chromophore binding and for transduction of conformational changes induced by light absorption by the chromophore to the effector domain of photoreceptors. Study of knotted proteins is of high importance for solving the fundamental problem of protein folding. We investigated the influence of chromophore and knot on the folding of the near-infrared iRFP developed from BphPs, RpBphP2. To this aim we obtained and characterized iRFP in its holoform (in complex with the chromophore) and its apoform (chromophore-free). The spectral features of iRFP in the apoform suggest that it retains the secondary and tertiary structures inherent to the holoform of protein. Apoprotein incorporates BV at a molar ratio of 1:1 as indicated by equilibrium microdialysis. The conformation and microenvironment of BV in the apo-iRFP/BV complex was similar to that for native iRFP. Denaturation – renaturation experiments on iRFP in holoform and apoform revealed that the knot does not prevent efficient protein folding while the chromophore stimulates protein aggregation and thus inhibits protein refolding.