Caratterizzazione Biofisica di Odorant Binding Proteins da Vertebrato e Possibili Applicazioni Biotecnologiche

Abstract

The Binding Protein family is a class of proteins characterized of a ligand binding site able to bind different probes of biological nature. For this reason it appears that these biomolecules are currently studied in several labs around the world since they also represent excellent candidates in designing of specific optical biosensors for detection of small analytes. Odorant Binding Proteins (OBPs), a sub-class of lipocalins, are defined by their property of reversibly binding volatile chemicals called “odorants”. Odorants and pheromones are important signals conditioning the behaviour of animals. These soluble proteins of low molecular mass, synthesized in the lateral nasal glands and secreted into the nasal mucosa, seem to play important roles in chemical communication [Pelosi P., Ann. N. Y.Acad. Sci., 1998]. They are present at high concentrations in biological fluids, involved in the perception and in the delivery of chemical messages of pheromonal significance. This class of proteins have been isolated from such as cow, rabbit, pig and mouse. Vertebrates' OBPs share a typical β-barrel folding and an hydrophobic binding pocket for ligands [Bianchet M.A., et al., Nat. Struct. Biol. 1996; Tegoni M., et al., Nat Struct Biol, 1996]. The subject of this PhD project is the structural and functional characterization of the eukaryotic Odorant Binding Proteins (OBPs) originally characterized from bovine and pig nasal mucosa. The work was based on recombinant OBPs obtained upon gene cloning in E. coli and protein expression and purification procedures. The main aim of the work was to perform a structural characterization of the OBPs by optical spectroscopy in order to investigate the stability and the conformational dynamics of these biomolecules. OBPs also represent an interesting and simple model for studying the phenomena of protein aggregation and how the formation of protein dimers and/or protein oligomers is depending upon single amino acid mutations in protein primary structure.

In addition, the project is aimed at acquiring basic knowledge on the structure and stability of proteins and at developing biotechnological applications such as the design of innovative optical OBP-based biosensors for the detection of small molecules. As a consequence, it is of interest to study the factors regulating the stability of these proteins, and to establish general guidelines that allow to address a large variety of different questions in biosensing applications. To this end, fluorescence correlation spectroscopy (FCS), fluorescence anisotropy measurements and ultracentrifugation (AUC) experiments were carried out to gain information on the thermodynamic parameters related to OBPs stability. The obtained results show that OBPs are good candidates as probe for specific optical biosensors for sensing small analytes. They also suggest their potential use as a biological scaffold for a variety of biotechnological applications. In fact, in the frame of this thesis project two different biosensor prototypes were realized: a) fluorescence-based biosensor; b) surface acoustic wave-based biosensor. In particular the fluorescence biosensors was realized by immobilizing the OBP onto carbon nano-tubes. The binding of the ligand, 1-octen-3-ol (OCT) to the immobilized OBP was detected following the efficiency of the process resonance energy transfer (RET) between intrinsic tryptophan residues of OBP and an energy acceptor 1-amino-anthracene (AMA) that fits in the OBP funnel. As regard the SAW-based biosensor, OBP were immobilized onto a wafer realized by deposition of 5 nm gold film on a 100 nm aluminum film and mounted on a TO39 package.

Both, fluorescence biosensors and surface acoustic wave (SAW) biosensors are currently being used in medicine, environment monitoring, biotechnology, food industry and security applications