ИСТИНА

A tremendous diversity of ligand binding proteins creates considerable opportunities for their scientific and medical applications [1]. A large group of odorant-binding proteins (OBPs) attracts high scientific interest as promising building blocks for construction of optical biosensors for dangerous substances such as toxic, explosives and so on. The important advantage of OBPs is the high structural plasticity of their binding site which allows engineering of artificial proteins with the same 3D structure but novel ligand specificities. [2]. It is important that sensitive element of any biosensor system should possess an essential stability to environmental influence that can be tested by protein stability to different denaturing agents. OBPs share a common nine-stranded β-barrel structure which encloses a ligand binding site composed of an internal cavity and an external loop scaffold. Despite an increasing number of studies on the stability and unfolding processes of proteins with β-barrel topology, these processes have been understood to a much lesser extent than for α- and α/β-proteins. Thus, investigation of proteins with β-barrel topology is of fundamental importance. Moreover, OBPs are interesting object for examination of the role of ligands in protein structure formation and stabilization. This work is focused on the study of dimeric bovine OBP (bOBP) which in contrast to classical OBPs is characterized by an unique folding pattern that involves the crossing of the α-helical domain of each monomer over the β-barrel of the other [3]. A recombinant bOBP was overproduced in bacterial system. The examination of unfolding – refolding of recombinant bOBP in the presence of guanidine hydrochloride (GdnHCl) by different spectroscopic methods and size-exclusion chromatography showed high denaturant resistance of the protein (mid point of unfolding is at more than 2 M GdnHCl). We also observed structural perturbation of bOBP at pre-denaturing concentrations of GdnHCl which should be considered if protein is used as sensitive element of biosensor system. The recombinant bOBP produced in bacterial system tends to accumulate in buffered solution in some state with features closed to native state of the protein. This state is stable and has decreased tendency for dimerization so as the protein exists as a mixure of monomeric and dimeric forms in buffered solution. The formation of native dimeric state of bOBP is promoted by an increasing of denaturant concentration up to 1.5 M GdnHCl. This process requires some reorganization of bOBP structure and goes through accumulation of an intermediate state. The revealed structural changes of bOBP at pre-denaturing concentrations of GdnHCl have a local character with overall structure of the protein being reserved and do not influence on the protein capability to bind ligand as showed by experiments on interaction with fluorescent ligand aminoanthracene-1. Thus, despite the complex energy landscape bOBP is stable enough for being used in biosensor systems.