ИСТИНА

The investigation of proteins with beta-barrel topology inherent to odorant-binding proteins (OBPs) is important fundamental task. OBPs also attract high scientific interest as they can be used at the construction of optical biosensors for dangerous substances, such as toxic and explosive molecules [1]. The bovine OBP (bOBP) folds into an unique dimeric form. Each monomer subunit of the bOBP possesses beta-barrel-like spatial structure which is typical of majority of OBPs. The beta-barrel of OBP is composed of eight beta strands and subsequent short alfa-helical segment that followed by the ninth beta-strand and the disordered C-terminal region of the protein. The dimeric molecule of bOBP is formed by crossing the α-helical domain from each monomer over the β-barrel of the other monomer [2]. This method of dimerization is termed as “domain swapping”. Here we studied the spectral properties of recombinant bOBP and its mutant forms bOBP-Gly121+ and GCC-bOBP using intrinsic UV and visible fluorescence and CD in the far and near UV region. It is believed that bOBP-Gly121+ and GCC-bOBP are not able to dimerize using the “domain swapping” and thus have monomeric form. The insertion of glycine after the residue in position 121 in bOBP (bOBP-Gly121+ mutant) allows the alfa-helix to fold back on the beta-barrel as results of the increased flexibility of the hinge connecting them. The W64C and H156C substitutions in GCC-bOBP restore the canonical disulfide bridge which is absent in bOBP and stabilizes the monomeric form of the mutant protein. Additionally we characterized the mutant proteins GCC-bOBP-W17F and GCC-bOBP-W133F bearing the single tryptophan residue. It was shown that all mutant forms preserve the secondary and tertiary structure inherent to monomeric recombinant bOBP though slight alterations can be observed. These small changes of structural organization of mutant proteins do not disrupt the ability of the proteins to bind its ligand as was tested by protein interaction with its fluorescent ligand aminoanthracene-1. This indicates that all studied proteins are functionally active. Among all mutant proteins GCC-bOBP has the secondary and tertiary structure that is the most close to recombinant bOBP. Joined analysis of fluorescent characteristics of GCC-bOBP-W17F and GCC-bOBP-W133F and peculiarities of tryptophan microenvironment of bOBP and GCC-bOBP allowed revealing a new factor which can contribute to protein fluorescent characteristics.