ABSTRACT Transmembrane proteins, such as G-protein-coupled receptors (GPCRs), are critical for many cellular functions. They are also the most popular drug targets for various diseases, including cancer. For both understanding cellular functions and drug development, it is necessary to measure their binding activities with molecular ligands and drug candidates. However, this has been a difficult task because of two challenges. First, transmembrane proteins are difficult to extract and purify, and they often lose their native conformations after isolation from the cellular membranes. Second, even if a membrane protein is successfully isolated, it remains challenging to measure its binding to ligands, especially with small molecule ligands. Small molecules comprise ~90% of the current drugs, but their binding kinetics cannot be easily measured with the existing detection technologies. This project addresses both challenges with a virion oscillator technology. Human GPCRs are displayed on the viral envelopes of human herpes simplex virus-1 (HSV-1), which removes the need of extraction, purification, and reconstitution of the transmembrane proteins. Each virion is then tethered to a sensor chip with a flexible polymer linker to form an oscillator. By applying an alternating electric field to the chip, the virion oscillates, and the oscillation amplitude is tracked in real-time with sub-nanometer precision using a plasmonic imaging technique. Upon binding of ligands or drugs to the GPCRs on the virion envelopes, the oscillation amplitude changes, from which binding kinetics and affinity are quantified. This project combines the virion display and microarray strengths at Johns Hopkins University, and plasmonic imaging and biosensing expertise at Arizona State University. The team has been working together and completed substantial preliminary experiments to demonstrate this new detection platform. The goal of this R33 project is to transform the technology into a powerful high-throughput platform for studying membrane proteins by 1) developing virion oscillator microarray chips (with 315 non-odorant human GPCRs on a single chip), 2) developing a plasmonic imaging system for high-throughput quantification of molecular binding kinetics, and 3) validating the virion oscillator microarray technology with cancer related GPCRs. It is anticipated that the virion oscillator detection technology will become a unique tool for studying cellular functions of membrane proteins, and quantifying binding of large and small molecule drugs with any types of membrane proteins.
