Quick characterization of binding onto unpurified GPCRs

In this technical note, we show how the Creoptix WAVE can be used for the kinetic analysis of G-protein binding onto detergent-solubilized, unpurified GPCRs. By shortening a typical 10-step purification process, this method requires significantly less material (30mL of cell culture) and less time (less than 1 hour), thereby offering an alternative for screening purposes at an early stage of the drug discovery process involving membrane proteins.

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 Creoptix WAVE 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 Creoptix WAVE 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-Go1, to a crude preparation of unpurified, n-dodecyl β-D-maltoside (DDM)-solubilized serotonin 5-HT1B receptor (5-HT1BR), combining the sample-buffer injection and a quick and “dirty” purification process, we show that the Creoptix WAVE delivers essential insights to GPCR signaling and pharmacology.

Materials and Methods

Trichoplusia ni insect cell membranes expressing the His-tagged 5-HT1BR, the agonist donitriptan and the engineered mini-Go1 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-HT1BR was then captured and amine-coupled onto a Ni2+-NTA chip (WAVEchip 4PCH-NTA). His-tagged mini-Go1 - 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-HT1BR is activated by the injection of the agonist donitriptan, prior to the binding of mini-Go1.

[Figure 1 TN200101-Creoptix-binding-unpurified-GPCRs.jpg] Figure 1 TN200101-Creoptix-binding-unpurified-GPCRs.jpg

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

[Figure 2 TN200101-Creoptix-binding-unpurified-GPCRs.jpg] Figure 2 TN200101-Creoptix-binding-unpurified-GPCRs.jpg

Figure 2: Sample buffer injection process

Results

Dose-response curves were recorded for 7 concentrations of mini-Go1 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-Go1 binding onto DDM-solubilized, donitriptan-bound 5-HT1BR. 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 KD values are in good agreement with previously reported fluorescent saturation binding assay (FSBA) data.1

[Figure 3 TN200101-Creoptix-binding-unpurified-GPCRs.jpg] Figure 3 TN200101-Creoptix-binding-unpurified-GPCRs.jpg

Figure 3: mini-Go1 kinetics

Key takeaways

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

  • 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

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