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.
Highlights
The problem of folding a protein molecule to a native functional structure is the hallmark of protein science
Our data obtained by gel filtration experiments, show that recombinant bovine OBP (bOBP) is a mixture of monomers and dimers under native conditions, and the full recombinant bOBP population is a dimer in the presence of 1.5 M guanidine hydrochloride (GdnHCl) (Fig. 6)
Such local alterations in the recombinant bOBP secondary and tertiary structure in a buffered solution are accompanied by a decreased capacity for recombinant bOBP dimerization in a buffered solution, which is a mixture of monomers and dimers (Fig. 6)
Summary
The problem of folding a protein molecule to a native functional structure is the hallmark of protein science. A large group of odorant-binding proteins (OBPs) in the lipocalin family share a common b-barrel structure with eight bstrands (residues 9–120) linked by a turn to a short a-helical domain (residues 124–141). The b-barrel in OBPs encloses a ligand binding site with an internal cavity formed by hydrophobic and aromatic amino acids as well as an external loop scaffold [7,8]. The proteins in this group are attractive as a tool in constructing optical biosensors for dangerous substances, such as toxic and explosive molecules [9,10,11]. The high structural plasticity of the OBP binding site allows to optimize the interactions of the protein with ligands that differ structurally from their cognate ligands [9,12,13]
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