ИСТИНА

An large group of odorant-binding proteins (OBPs) attract high scientific interest as promising building blocks for construction of optical biosensors for dangerous substances such as toxic, explosive molecules and so on. OBPs share a common nine-stranded β-barrel structure which encloses a ligand binding site composed of both an internal cavity and an external loop scaffold. Despite an increasing number of studies on the stability and unfolding-refolding processes of protein with -barrel topology, these processes have been understood to a much lesser extent than for - and /- proteins. Thus, investigation of proteins with β-barrel topology is of high fundamental importance. Moreover, using OBPs as an object of investigations gives an opportunity of studing the role of ligand in the structure and in the stabilization of proteins in their native state. This work is focused on the study of triple mutant form of bovine odorant-binding protein GCC-bOBP (Gly 122+, W64C, H156C) having monomeric nature typical among other mammalian OBPs. Insertion of Gly after residue in the 121 position is sufficient for restoring of monomeric state of bOBP as previously proved [1]. Still, introducing of common among other OBPs disulfide bridge is required for stabilizing of monomeric protein [2]. The investigation of unfolding – refolding of GCC-bOBP in the presence of guanidine hydrochloride (GdnHCl) by spectroscopic methods was carried out. The unfolding – refolding equilibrium is reached after 24 h of the protein incubation at desired denaturant concentrations. GCC-bOBP is highly resistant to denaturing action of GdnHCl with mid point of unfolding at more than 2 M GdnHCl. This value are characteristic for other monomeric OBPs but are in some discrepancy with previously reported mid point value of 2.9 M GdnHCl for GCC-bOBP [3]. But these data were calculated from GCC-bOBP unfolding curves measured after 2 h of protein incubation in the presence of GdnHCl which is far from equilibration. In the folding pathway of GCC-bOBP two intermediate states are firstly observed at the moderate denaturant concentrations proceeded by the full unfolding of the protein. These states of GCC-bOBP accumulating at pre-denaturing GdnHCl concentrations are structurally close to native state of the protein. The possible effect of these structural changes of GCC-bOBP on the protein capability to bind ligand is discussed.