Structure and stability of recombinant bovine odorant-binding protein: II. Unfolding of the monomeric forms.

Olga V Stepanenko,Konstantin K Turoverov,Irina M Kuznetsova,Vladimir N Uversky,Olesya V Stepanenko,Denis O Roginskii

doi:10.7717/peerj.1574

Abstract

In a family of monomeric odorant-binding proteins (OBPs), bovine OBP (bOBP), that lacks conserved disulfide bond found in other OBPs, occupies unique niche because of its ability to form domain-swapped dimers. In this study, we analyzed conformational stabilities of the recombinant bOBP and its monomeric variants, the bOBP-Gly121+ mutant containing an additional glycine residue after the residue 121 of the bOBP, and the GCC-bOBP mutant obtained from the bOBP-Gly121+ form by introduction of the Trp64Cys/His155Cys double mutation to restore the canonical disulfide bond. We also analyzed the effect of the natural ligand binding on the conformational stabilities of these bOBP variants. Our data are consistent with the conclusion that the unfolding-refolding pathways of the recombinant bOBP and its mutant monomeric forms bOBP-Gly121+ and GCC-bOBP are similar and do not depend on the oligomeric status of the protein. This clearly shows that the information on the unfolding-refolding mechanism is encoded in the structure of the bOBP monomers. However, the process of the bOBP unfolding is significantly complicated by the formation of the domain-swapped dimer, and the rates of the unfolding-refolding reactions essentially depend on the conditions in which the protein is located.

Highlights

Odorant binding proteins (OBPs) are important components of olfactory apparatus in vertebrates where they play a specific role in olfaction by interacting directly with odorants (Xu et al, 2005)
We analyzed how introduced mutations affected the structural flexibility of bovine OBP (bOBP) utilizing the power of the FlexPred tool that rapidly predicts absolute per-residue fluctuations from a threedimensional structure of a query protein (Jamroz, Kolinski & Kihara, 2012)
Since the FlexPred tool provides real-value fluctuations of globular proteins based on their static structures by considering Cα atoms contact number and known B-factor values, the fact that these three proteins, bOBP (PDB ID: 1OBP), GCC-bOBP (PDB ID: 2HLV), and porcine OBP (pOBP) (PDB ID: 1A3Y), are predicted to have similar structural flexibility indicates that mutations introduced to generate the monomeric GCC-bOBP or present in the naturally monomeric pOBP do not dramatically affect Cα atoms contact number and B-factors of the resulting structures

Summary

Introduction

Odorant binding proteins (OBPs) are important components of olfactory apparatus in vertebrates where they play a specific role in olfaction by interacting directly with odorants (Xu et al, 2005). Analysis of the bOBP/OCT refolding from the completely unfolded state revealed that the dependencies of various structural characteristics of the bOBP/OCT on GdnHCl concentrations depend on the incubation time of this complex in the presence of the denaturant (see Fig. 3).

Results

Conclusion