Structure and stability of recombinant bovine odorant-binding protein: I. Design and analysis of monomeric mutants
- Published
- Accepted
- Subject Areas
- Biochemistry, Biophysics, Molecular Biology
- Keywords
- odorant-binding protein, fluorescence, protein, domain swapping, chemical denaturation, circular dichroism
- Copyright
- © 2015 Stepanenko et al.
- Licence
- This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, reproduction and adaptation in any medium and for any purpose provided that it is properly attributed. For attribution, the original author(s), title, publication source (PeerJ PrePrints) and either DOI or URL of the article must be cited.
- Cite this article
- 2015. Structure and stability of recombinant bovine odorant-binding protein: I. Design and analysis of monomeric mutants. PeerJ PrePrints 3:e1445v1 https://doi.org/10.7287/peerj.preprints.1445v1
Abstract
Bovine odorant-binding protein (bOBP) differs from other lipocalins by lacking the conserved disulfide bond and 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.
Author Comment
This manuscript is a submission to PeerJ. It opens a series of articles dedicated to the analysis of the effect of the environmental feature (including the presence of crowding agents) on structural properties and conformational stability of bOBP.