Quick characterization of binding onto unpurified GPCRs

Summary

G protein-coupled receptors (GPCRs) are important therapeutic targets due to their essential role in regulating physiological processes. Being the target of over 30% of approved drugs, GPCRs are notoriously difficult to study because their hydrophobicity makes them extremely unstable following extraction from the cell membrane.

With patented no-clog microfluidics, the WAVEsystem, a Creoptix technology, allows researchers to investigate GPCRs both in solution (detergent-solubilized or reconstituted into a lipidic environment such as nanodiscs) and in membranes, where they can retain their native conformation. Using a proprietary Grating-Coupled  Interferometry (GCI) technology to deliver exceptional sensitivity, the WAVEsystem can resolve a wide range of affinities and off-rates for enhanced understanding of G-protein/GPCR interactions.

By characterizing the binding kinetics and affinity of the G-protein, mini-G_o1, to a crude preparation of unpurified, n-dodecyl β-D-maltoside (DDM)-solubilized serotonin 5-HT_1B receptor (5-HT_1BR), combining the sample-buffer injection and a quick and “dirty” purification process, we show that the WAVEsystem delivers essential insights to GPCR signaling and pharmacology.

Materials and Methods

Trichoplusia ni insect cell membranes expressing the His-tagged 5-HT _1B R, the agonist donitriptan and the engineered mini-G _o1 were kindly provided by Dr. Chris Tate (MRC Laboratory of Molecular Biology - Cambridge, UK).

Cell membranes from ~ 30 mL of culture were solubilized for 30 min with 0.5% DDM and the crude solubilizate was recovered in the supernatant after a 30 min centrifugation at 20,000 xg. The proposed quick and “dirty” procedure takes less than 1h and it is suitable for stable GPCRs with expression levels of at least 0.5 mg/L of cell culture. Details of the quick and “dirty” purification process are shown in Figure 1.

His-tagged, DDM-solubilized 5-HT_1BR was then captured and amine-coupled onto a Ni²⁺-NTA chip (WAVEchip 4PCH-NTA). His-tagged mini-G_o1 - the negative control - was captured and amine-coupled onto the reference channel of the WAVEchip. G-protein binding onto solubilized GPCR was monitored using the sample-buffer injection process (Figure 2), where 5-HT_1BR is activated by the injection of the agonist donitriptan, prior to the binding of mini-G_o1.

Figure 1: Standard vs. quick and "dirty" GPCR purification process

Figure 2: Sample buffer injection process

Results

Dose-response curves were recorded for 7 concentrations of mini-G_o1 ranging from 1 nM to 16.8 μM in sample buffer. Flow rate was 30 μL/min and measurements were carried out at 10°C.

Figure 3 shows the binding curves recorded for mini-G_o1 binding onto DDM-solubilized, donitriptan-bound 5-HT_1BR. Data were double-referenced and fitted with a heterogenous ligand model (accounting for two ligand species). Kinetic parameters are shown in the table below, and K_D values are in good agreement with previously reported fluorescent saturation binding assay (FSBA) data.¹

Figure 3: mini-G_o1 kinetics

Key takeaways

Understand GPCR signaling and pharmacology, and speed up your early stage drug discovery process with the WAVEsystem:

• Use less material: confidently work with unpurified solubilizates
• Save time: work with suboptimal assay conditions
• WAVE goodbye to lengthy and laborious receptor purification procedures: welcome contaminants and work with crude samples with no-clog, robust microfluidics

Great for:

• Screening of different G proteins binding onto the same receptor
• Screening of G protein binding in different detergents
• Drug characterization (inverse agonist, agonist) with G protein binding as a read-out

References

1. Nehmé R, Carpenter B, Singhal A, Strege A, Edwards PC, White CF, et al. (2017) Mini-G proteins: Novel tools for studying GPCRs in their active conformation. PLoS 12(4): e0175642.: https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0175642