| Insects rely on smell in every aspect of their lives. Pheromones are chemicals produced by a species to influence the behavior of the same species. In the fruit fly Drosophila melanogaster, one volatile pheromone is known: 11-cis-vaccenyl acetate (cVA). cVA is required for both attraction and sexual behaviors.;The odorant binding protein (OBP) LUSH is required for cVA perception. When LUSH is knocked out, not only does the fly lose attraction to cVA, but the neurons that detect cVA become inactive, even losing their normal spontaneous activity. This activity is restored by re-addition of LUSH, but not other OBPs. This indicates that LUSH is the activating factor to these neurons.;The X-ray crystal structure of LUSH bound to cVA was solved. This allowed identification of crucial contacts between cVA and phenylalanine F121 of LUSH (F121) that changed the conformation of LUSH. Mutation of F121 to alanine (F121A-LUSH) resulted in not only lowered capacity to confer cVA perception, but also lowered spontaneous activity. Mutation of a second residue, aspartic acid 118, to alanine (D118-LUSH), resulted in much higher spontaneous activity that wild-type LUSH. These results indicate that LUSH is the activating factor in cVA perception. We solved the X-ray crystal structure of both these mutants, which suggested reasons for each protein's phenotype. We also determined the affinity of each protein for cVA and compounds with similar structures ("cVA analogs"), in order to determine if the in vivo activity could be explained by specificity of LUSH and the mutant proteins for cVA. The mutant proteins displayed only slight reductions in affinity for cVA. Wild-type LUSH, however, binds to some of the cVA analogs with higher affinity than the native pheromone. Finally, we undertook NMR studies to determine the effect that cVA has on the solution conformation of LUSH and the mutant proteins. Any change in the structure of the pheromone or protein significantly changed the solution conformation of LUSH. We identified surface residues that possibly interact with olfactory receptors to induce activity. Finally, we proposed a model of the mechanism of cVA perception as mediated by LUSH. |
