Abstract
Bovine odorant-binding protein (bOBP) differs from other lipocalins by lacking the conserved disulfide bond and for being able to form the domain-swapped dimers. To identify structural features responsible for the formation of the bOBP unique dimeric structure and to understand the role of the domain swapping on maintaining the native structure of the protein, structural properties of the recombinant wild type bOBP and its mutant that cannot dimerize via the domain swapping were analyzed. We also looked at the effect of the disulfide bond by designing a monomeric bOBPs with restored disulfide bond which is conserved in other lipocalins. Finally, to understand which features in the microenvironment of the bOBP tryptophan residues play a role in the defining peculiarities of the intrinsic fluorescence of this protein we designed and investigated single-tryptophan mutants of the monomeric bOBP. Our analysis revealed that the insertion of the glycine after the residue 121 of the bOBP prevents domain swapping and generates a stable monomeric protein bOBP-Gly121+. We also show that the restored disulfide bond in the GCC-bOBP mutant leads to the noticeable stabilization of the monomeric structure. Structural and functional analysis revealed that none of the amino acid substitutions introduced to the bOBP affected functional activity of the protein and that the ligand binding leads to the formation of a more compact and stable state of the recombinant bOBP and its mutant monomeric forms. Finally, analysis of the single-tryptophan mutants of the monomeric bOBP gave us a unique possibility to find peculiarities of the microenvironment of tryptophan residues which were not previously described.
Highlights
Lipocalins constitute a family of carrier proteins that transport various small hydrophobic molecules ranging from lipids to retinoids, steroids, and bilins
These analyses revealed that the wild type Bovine odorant-binding protein (bOBP) and its four mutants are expected to be rather ordered and that mutations do not induce significant changes in the bOBP disorder propensity
We show here that the insertion of Gly121+ leads to disruption of the domain swapping mechanism, resulting in a stable monomeric mutant protein bOBP-Gly121+
Summary
Lipocalins constitute a family of carrier proteins that transport various small hydrophobic molecules ranging from lipids to retinoids, steroids, and bilins. Classic odorant binding protein (OBP) is characterized by a specific monomeric fold, where the eight β-strands, a short α-helical region, and the ninth β-strand interact to form a β-barrel followed by the disordered C-terminal tail (Bianchet et al, 1996; Flower, North & Sansom, 2000). In the bOBP dimer, each of the two protomers forms a β-barrel that interacts with the α-helical portion of the C-terminal tail of other protomer via the domain swapping mechanism. Such a mechanism was described for many dimeric and oligomeric protein complexes, where it plays important structural and functional roles (Bennett, Schlunegger & Eisenberg, (1995); Van der Wel, (2012)). GCC-bOBP-W133F, each containing a single tryptophan residue, for the characterization of the specific features of the microenvironments of these tryptophan residues that affect the intrinsic fluorescence characteristics of this protein
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