UC Berkeley

Abstract

Engineering of Olfactory Receptor OlfCc1 for Directed Ligand Sensitivity

by

Allison Paige Berke

Joint Doctor of Philosophy

with the University of California San Francisco

University of California, Berkeley

Professor Song Li, Chair

Due to structural similarity, OlfCc1and its mammalian analogue V2R2 are hypothesized to respond to amino acid ligands in a calcium-mediated fashion. By analyzing receptor structure and making targeted mutations, the specificity and sensitivity of the receptor should be tunable, within the range of OR response thresholds. OlfCc1 responds to the amino acids isoleucine and leucine, in a calcium-dependent manner. V2R2 shows a similar response profile, with additional bimodal responses to valine. Additionally, both function as amine receptors, responding to isoamylamine and 2-methylbutylamine. This represents a TAAR-independent amine-sending pathway. Targeted mutations to OlfCc1 have successfully altered the sensitivity of the receptor, through mutating residues in the proximal pocket that are predicted to be three Angstroms or less from the docked ligand. Additional docking with calcium in the binding pocket has clarified distal pocket residues that coordinate the side chain of the amino acid ligand. Mutations to these residues have successfully altered the specificity of the receptor, including mutations to align its binding profile with that of the calcium-sensing receptor. Residues affecting amine-group binding as well as side-chain stabilization and calcium binding have been identified through modeling and confirmed through expression and functional testing in HEK cells.

The utility of and interest in these findings have both engineering and biological significance. Being able to target receptor functionality through directed modeling and mutagenesis opens up the olfactory receptors, naturally occurring and highly sensitive native sensing elements, as a family of candidate in vitro sensing elements. Their ability to be tuned along a gradient of concentration-dependent responses (showing EC50 tunability) indicates that they may be useful as reporter elements. Additionally, the ability to make verifiable and accurate predictions about receptor functionality from an in silico model is useful to the study of many receptors. The utility of using OlfCc1 and V2R2 in particular stems from their unique expression profiles (expressed near-ubiquitously in certain populations of olfactory and vomeronasal neurons) and their close homology with LIVBP, MGluR, and PBP proteins. Showing through modeling and mutagenesis how OR regions of similarity correspond to binding profiles gives us insight into how receptors function combinatorially.