The Quaternary Structure of the Recombinant Bovine Odorant-Binding Protein Is Modulated by Chemical Denaturants

Abstract

A large group of odorant-binding proteins (OBPs) has attracted great scientific interest as promising building blocks in constructing optical biosensors for dangerous substances, such as toxic and explosive molecules. Native tissue-extracted bovine OBP (bOBP) has a unique dimer folding pattern that involves crossing the α-helical domain in each monomer over the other monomer’s β-barrel. In contrast, recombinant bOBP maintaining the high level of stability inherent to native tissue bOBP is produced in a stable native-like state with a decreased tendency for dimerization and is a mixture of monomers and dimers in a buffered solution. This work is focused on the study of the quaternary structure and the folding-unfolding processes of the recombinant bOBP in the absence and in the presence of guanidine hydrochloride (GdnHCl). Our results show that the recombinant bOBP native dimer is only formed at elevated GdnHCl concentrations (1.5 M). This process requires re-organizing the protein structure by progressing through the formation of an intermediate state. The bOBP dimerization process appears to be irreversible and it occurs before the protein unfolds. Though the observed structural changes for recombinant bOBP at pre-denaturing GdnHCl concentrations show a local character and the overall protein structure is maintained, such changes should be considered where the protein is used as a sensitive element in a biosensor system.

Structure and stability of recombinant bovine odorant-binding protein: III. Peculiarities of the wild type bOBP unfolding in crowded milieu

“…This high conformational stability is indicated by the fact that the recombinant bOBP unfolding is characterized by the halftransition point of > 2 M GdnHCl (Stepanenko et al 2014b). We have also established that the unfolding of the recombinant bOBP is a completely reversible process, whereas the preceding process of its dimerization is the irreversible event (Stepanenko et al 2014b).…”

“…Despite its disturbed fold, the recombinant bOBP is characterized by high conformational stability, which is comparable with that of the native (isolated from tissue) bOBP (Mazzini et al 2002), pOBP (Staiano et al 2007;Stepanenko et al 2008), and other -rich proteins (Stepanenko et al 2012;Stepanenko et al 2013;Stepanenko et al 2014a). This high conformational stability is indicated by the fact that the recombinant bOBP unfolding is characterized by the halftransition point of > 2 M GdnHCl (Stepanenko et al 2014b). We have also established that the unfolding of the recombinant bOBP is a completely reversible process, whereas the preceding process of its dimerization is the irreversible event (Stepanenko et al 2014b).…”

“…Our previous studies revealed that there is a noticeable difference between the recombinant bOBP and a natural form of this protein isolated from tissues (Stepanenko et al 2014b). Here, recombinant bOBP forms a stable native-like conformation with the decreased dimerization potential and therefore exists as a mixture of monomeric and dimeric variants (Stepanenko et al 2014b). It is likely that the formation of the domain-swapped dimer by the bOBP represents a complex process that requires particular organization of the secondary and tertiary structures of the bOBP monomers.…”

“…It is likely that the formation of the domain-swapped dimer by the bOBP represents a complex process that requires particular organization of the secondary and tertiary structures of the bOBP monomers. We hypothesized that the recombinant bOBP has perturbed packing of its α-helical region and some β-strands, and that these perturbations in packing of the secondary structure elements might affect the formation of native domain-swapped dimer (Stepanenko et al 2014b).…”

“…This process requires noticeable reorganization of the bOBP structure and is accompanied by the formation of a stable, more compact intermediate state which is maximally populated at 0.5 M GdnHCl. Cooperative unfolding of the recombinant bOBP is induced by the increase of the GdnHCl concentration above 1.5 M, whereas this protein is completed by ~3M GdnHCl (Stepanenko et al 2014b).…”