Characterization of a Deswapped Triple Mutant Bovine Odorant Binding Protein

Eugenia Polverini,Roberto Ramoni,Robert T Sorbi,Alberto Mazzini,Conti Virna,Paolo Lardi,Roberto Favilla

doi:10.3390/ijms12042294

Abstract

The stability and functionality of GCC-bOBP, a monomeric triple mutant of bovine odorant binding protein, was investigated, in the presence of denaturant and in acidic pH conditions, by both protein and 1-aminoanthracene ligand fluorescence measurements, and compared to that of both bovine and porcine wild type homologues. Complete reversibility of unfolding was observed, though refolding was characterized by hysteresis. Molecular dynamics simulations, performed to detect possible structural changes of the monomeric scaffold related to the presence of the ligand, pointed out the stability of the β-barrel lipocalin scaffold.

Highlights

OBPs belong to the kernel lipocalin family [1,2], which, despite the low degree of sequence similarity among its members, is characterized by a well conserved eight-stranded antiparallel β-barrel [3,4]
The fluorescence spectra of the three proteins, collected under identical conditions differ in intensity, but have a similar shape
Assuming that the homologous Trp residues have the same fluorescence quantum yield, independently of the protein they belong to, as suggested by their very similar environments in the crystals, the relative contribution of each Trp residue to the total fluorescence can be estimated by comparing the total fluorescence intensity of each protein, collected under identical conditions (Table 1)

Summary

Introduction

OBPs belong to the kernel lipocalin family (a member of the calycin superfamily) [1,2], which, despite the low degree of sequence similarity among its members, is characterized by a well conserved eight-stranded antiparallel β-barrel [3,4]. The study of protein structure-function relationships has been largely facilitated by the development of site directed mutagenesis, that offers the possibility to modify the sequence of any protein at will and to understand, at least in principle, the role played by the mutated residues from their effect on the structural and functional properties of that protein [9] This strategy was applied to bovine odorant binding protein (bOBP), a swapped dimeric protein [10,11], to turn it into a functional monomer at neutral pH. Two modifications were made to bOBP: first, a Gly residue was inserted after

Objectives

Results

Conclusion