Defining the chemical space for ligands of odorant - binding proteins

Lead Research Organisation: Rothamsted Research
Department Name: Unlisted

Abstract

We will investigate interactions between olfactory ligands of Drosophila melanogaster and odorant-binding proteins (OBPs) involved in their peripheral perception. Objectives:1) Construction of libraries of known ligands and interaction with OBPs:Libraries of known semiochemicals for D. melanogaster will be brought into contact with each recombinant OBP. Interactions will be monitored by determining which compounds are removed from the libraries. 2) Production of recombinant OBPs:D. melanogaster genes encoding putative OBPs will be screened for expression in antennae by RT-PCR. The selected cDNAs will be expressed into recombinant proteins and the proteins purified. 3) Testing of natural metabolites for interactions with OBPs:To complete the list of semiochemicals studied, we will screen collections of other compounds relating to insect/insect interactions or attraction to environmental cues. 4) Definition of chemical space for semiochemicals to bind to OBPs:A small number of OBPs that bind either one or a subset of ligands will be interacted with synthetic analogues to describe the chemical space required for OBP interaction. 5) Use of spectroscopic and other physical systems for confirming ligand-protein interactions:Ligands labelled with either stable isotopes (13C, 2H) or fluorine will be interacted with OBPs to determine whether slow or fast exchange binding occurs in solution. Nuclear overhauser coupling experiments will determine features of the binding site. 6) NMR spectroscopic identification of binding site amino acids:Recombinant OBPs expressed in the presence of 15N will allow detection of the OBP by 15N/1H HSQC 2D and determination of the amino acids involved in binding. 7) Design of novel ligands and identification of anthropogenic ligands binding to a particular OBP:The definition of chemical space for OBPs will allow the design of chemicals that act as suicide or alternative ligands for development in pest control and biosensors.

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