Determining the binding capability of the mouse major urinary proteins using 2-naphthol as a fluorescent probe.
The mouse major urinary proteins (MUPs) are an ensemble of isoforms secreted by adult male mice and involved in sexual olfactory communication. MUPs belong to the lipocalin superfamily, whose conserved structure is a beta-barrel made of eight antiparallel beta-strands forming a hydrophobic pocket that accommodates small organic molecules. A detailed knowledge of the molecular mechanism associated to the binding of those molecules can guide protein engineering to devise mutated proteins where the ligand specificity, binding affinity, and release rate can be modulated. Proteins with such peculiar properties may have interesting biotechnological applications for pest control, as well as in food and cosmetic industries. In this work, we demonstrate that the fluorescent molecule 2-naphthol binds to the natural ligand's binding site of MUPs with high affinity. In addition, we show that 2-naphthol binds to MUPs in its protonated form, that its fluorescence is blue-shifted, and the quantum yield is increased, thus confirming the high hydrophobicity of the protein pocket and the absence of proton acceptors inside the binding site. At large the results presented, besides demonstrating that the use of 2-naphthol provides a convenient and quick method for testing MUPs binding activity and to ascertain the quality of the protein preparation, suggest that MUPs can represent an interesting system for studying the photophysical characteristics of fluorescent molecules in a highly hydrophobic environment.
Sartor
G
G
Department of Experimental and Evolutionary Biology, University of Bologna, Bologna, Italy.
Pagani
R
R
Ferrari
E
E
Sorbi
R T
RT
Cavaggioni
A
A
Cavatorta
P
P
Spisni
A
A
D000818:Animals; D001667:Binding, Competitive; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry*; D051379:Mice; D009284:Naphthols / Q000737:chemistry*; D011506:Proteins / Q000032:analysis*
D005456:Fluorescent Dyes; D009284:Naphthols; D011506:Proteins; C035737:major urinary proteins; C028405:2-naphthol