G protein coupled receptors (GPCRs) are ubiquitous signal transducers, highly sought after as pharmaceutical targets. Currently, half of all marketed therapeutics target GPCRs, and in many cases the off-target effects of these pharmaceutical products are also mediated through GPCRs. Due to their inherent instability and insolubility GPCRs are difficult to study;however, their essential role in biological processes certainly establishes this as a compelling and worthwhile pursuit. Lipid bilayer nanodiscs have emerged as a convenient format by which to solubilize membrane-bound proteins, mimicking their native environment while simultaneously stabilizing these molecules. Solubilizing GPCRs within the nanodisc construct necessitates the development of appropriate analytical assays capable of screening the ligand binding of multiple receptors and receptor subtypes. Silicon photonic devices such as optical microring resonators are highly multiplexable devices that are sensitive to small changes in refractive index near the ring surface. Biomolecules can be covalently attached to microrings and subsequent binding events can be characterized by monitoring each of the individually addressable microrings. This methodology offers the additional advantage of small sample volume requirements. By immobilizing nanodisc-solubilized ? adrenergic receptors onto the microring resonator platform, we aim to investigate G protein coupling and ? arrestin binding of these receptors in response to both agonists and antagonists. The effects of receptor dimerization will also be examined. By combining the nanodisc and microring resonator technologies, bioassays will be developed to characterize the effects of various ligands on GPCR binding and off-target effects in a highly multiplexable platform.
Approximately half of currently marketed pharmaceuticals target G protein coupled receptors (GPCRs). There is a significant need for highly multiplexed analysis platforms to characterize G protein coupling and arrestin binding of these receptors as indicators of potential off-target drug effects while maintaining the receptors in a native-like liid bilayer environment. In the proposed work, nanodisc-solubilized adrenergic receptors will be immobilized on silicon optical microring resonator platforms to investigate receptor binding in response to various ligands.
|Sloan, Courtney D Kuhnline; Marty, Michael T; Sligar, Stephen G et al. (2013) Interfacing lipid bilayer nanodiscs and silicon photonic sensor arrays for multiplexed protein-lipid and protein-membrane protein interaction screening. Anal Chem 85:2970-6|
|Marty, Michael T; Sloan, Courtney D Kuhnline; Bailey, Ryan C et al. (2012) Nonlinear analyte concentration gradients for one-step kinetic analysis employing optical microring resonators. Anal Chem 84:5556-64|