ИСТИНА

A study of folding of the proteins with beta-barrel topology is an important fundamental task. Members of a large group of odorant-binding proteins (OBPs) possess a beta-barrel-like spatial structure. Classical OBPs show a nine-stranded β-barrel structure which encloses a ligand binding site composed of both an internal cavity and an external loop scaffold [1]. The possibility of an adjustment of the OBP binding site on the interactions of the protein with non-native ligands due to its high structural plasticity makes the proteins of this group highly attractive at the construction of optical biosensors for dangerous substances, such as toxic and explosive molecules [2]. Additionally, OBPs represents an interesting object for examination of the role of ligands in protein structure formation and stabilization. The bovine OBP (bOBP) has an unique dimeric folding pattern in contrast to classical monomeric form of the majority of OBPs [3]. The dimerization of bOBP is induced by “domain swapping” which involves crossing the α-helical domain from each monomer over the β-barrel of the other monomer. The “domain swapping” is widespread in many proteins greatly affecting their structure and functions. Moreover “domain swapping” is involved in amyloid fibril formation. In this work we focused on the investigation of guanidine hydrochloride (GdnHCl) induced unfolding – refolding of recombinant bOBP and the influence of “domain swapping” mechanism of dimerization on beta-barrel stability and the folding pathway. Showing high resistance to denaturing action of GdnHCl the recombinant bOBP undergoes some structure reorganization at pre-denaturing concentrations of GdnHCl. These structural changes have local character and do not affect the protein capability to bind ligand but allow the recombinant bOBP to acquire the native dimeric form. In contract to folding of the recombinant bOBP these dimerization of the protein appears to be irreversible. The obtained data were compared with the results of the study of stability and folding pathway of tissue-extracted bOBP and two mutant forms of bOBP which are not able to form dimeric molecule using the “domain swapping” mechanism. All studied proteins show significant stabilization of the structure against denaturing action at a formation of a complex with the native ligand 1-Octen-3-ol.