Binding of polycyclic aromatic hydrocarbons to mutants of odorant-binding protein: a first step towards biosensors for environmental monitoring.
Polycyclic aromatic hydrocarbons are among the most threatening pollutants widely present in the environment. Simple and economic methods of continuous monitoring of these compounds in real time are not yet available, although becoming increasingly needed. Odorant-binding proteins (OBPs) present unique characteristics of thermal and chemical stability for building robust, reliable, and inexpensive biosensors for such molecules. To investigate this possibility, we have engineered the pig OBP, whose three-dimensional structure has been resolved, introducing a tryptophan residue in the core of the binding pocket, as a fluorescence reporter for the presence of bound ligands. Binding affinities of several polyaromatic hydrocarbons to mutagenically modified OBPs were measured in competitive binding assays. Moreover, the presence of aromatic ligands was also successfully monitored in the modified OBPs by recording the quenching of intrinsic fluorescence of the protein. These data indicate that OBPs bind several aromatic polycyclic compounds with good affinities, that the specificity of these proteins can be easily modified by changing specific amino acid residues and that the introduction of a tryptophan residue in the binding site allows monitoring of aromatic ligands using direct fluorescence measurements.
Wei
Yin
Y
Dipartimento di Chimica e Biotecnologie Agrarie, University of Pisa, Italy.
Brandazza
Anna
A
Pelosi
Paolo
P
D000818:Animals; D001665:Binding Sites / Q000235:genetics; D015374:Biosensing Techniques; D004784:Environmental Monitoring / Q000379:methods; D008958:Models, Molecular; D009154:Mutation; D011084:Polycyclic Aromatic Hydrocarbons / Q000378:metabolism*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D018035:Receptors, Odorant / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013552:Swine
D011084:Polycyclic Aromatic Hydrocarbons; D018035:Receptors, Odorant; C045648:odorant-binding protein