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. 2012;7(9):e44911.
doi: 10.1371/journal.pone.0044911. Epub 2012 Sep 28.

Characterization of de novo synthesized GPCRs supported in nanolipoprotein discs

Tingjuan Gao  1 , Jitka Petrlova, Wei He, Thomas Huser, Wieslaw Kudlick, John Voss, Matthew A Coleman
Affiliations

Characterization of de novo synthesized GPCRs supported in nanolipoprotein discs

Tingjuan Gao et al. PLoS One. 2012.

Abstract

The protein family known as G-protein coupled receptors (GPCRs) comprises an important class of membrane-associated proteins, which remains a difficult family of proteins to characterize because their function requires a native-like lipid membrane environment. This paper focuses on applying a single step method leading to the formation of nanolipoprotein particles (NLPs) capable of solubilizing functional GPCRs for biophysical characterization. NLPs were used to demonstrate increased solubility for multiple GPCRs such as the Neurokinin 1 Receptor (NK1R), the Adrenergic Receptor â2 (ADRB2) and the Dopamine Receptor D1 (DRD1). All three GPCRs showed affinity for their specific ligands using a simple dot blot assay. The NK1R was characterized in greater detail to demonstrate correct folding of the ligand pocket with nanomolar specificity. Electron paramagnetic resonance (EPR) spectroscopy validated the correct folding of the NK1R binding pocket for Substance P (SP). Fluorescence correlation spectroscopy (FCS) was used to identify SP-bound NK1R-containing NLPs and measure their dissociation rate in an aqueous environment. The dissociation constant was found to be 83 nM and was consistent with dot blot assays. This study represents a unique combinational approach involving the single step de novo production of a functional GPCR combined with biophysical techniques to demonstrate receptor association with the NLPs and binding affinity to specific ligands. Such a combined approach provides a novel path forward to screen and characterize GPCRs for drug discovery as well as structural studies outside of the complex cellular environment.

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Conflict of interest statement

Competing Interests: Dr. Kudlicki is employed by Life Technologies. Life Technologies, (5791 Van Allen Way, Carlsbad), provided funding through the University of California Discovery Grant program. As a funder they had no role in study design, data collection and analysis, decision topublish, or preparation of the manuscript. They do have a financial interest through the marketing and sale of cell-free technologies. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. Co-expression with NLPs increased solubility for NK1R, ADRB2, and DRD1 and retained their activity.
(a) Comparison of GPCR solubility when expressed with or without NLPs. NK1R, ADRB2, and DRD1 were expressed as GFP fusion proteins. (b) The dot blot of GPCR-NLPs when incubating with (i) 100 nM fluorescent-labeled ligand and (ii) 100 nM fluorescent-labeled ligand and 100 µM non-labeled ligand. (iii) Non-specific signal of 100 nM fluorescent-labeled ligand alone on the filter paper as the control. The tests were done with 3 replicates for NK1R, 2 replicates for ADRB2 and 2 replicates for DRD1. (c) Quantification of dot blot assay. The significant difference of fluorescence intensity between (i) and (ii), (iii) indicates activity of NK1R, ADRB2 and DRD1.
Figure 2
Figure 2. Diffusion curves of lipid vesicles, NK1R and NK1R-NLP complexes.
The lipids and NK1R were labeled by Texas Red and GFP respectively. The cross correlation of Texas Red and GFP represents the interaction between lipids and NK1R, indicating the formation of NK1R-associated NLPs. The diffusion times of lipid vesicles, NK1R and NK1R- NLP complexes are 4.46, 0.17, and 0.51 ms respectively.
Figure 3
Figure 3. EPR spectra of 4-TOAC SP are sensitive to the presence of NLPs containing NK1R.
4-TOAC SP in buffer or combined with bR-containing NLP gave a narrower spectrum (h−1/h−0 ∼0.44). The spectrum for 4-TOAC SP was significantly broadened when NK1R-NLP complexes were present (red line, h−1/h0 = 0.34) reflecting the diminished rotational freedom of its receptor-bound state.
Figure 4
Figure 4. Saturation binding assay of FAM-SP on filter paper after interacting with NK1R-NLPs.
The fluorescence intensity was averaged through 3 replicates with the error bar showing the standard deviation. Each data point represented intensity of different amounts of FAM-SP interacting with NK1R-NLPs subtracted by non-specific adsorption of the same amounts of FAM-SP to the paper. The solid curve represents specific binding. The dashed curve represents non-specific binding (NK1R-NLPs saturated by excessive amount of non-labeled SP). The binding curve was fit to an “OneSiteBind” model Y  =  Bmax × X/(Kd + X), where Y represents fluorescence intensity caused by binding and X represents concentration of FAM-SP in the solution after reaction. The fitting results gave (3.5±0.3) ×106 (fluorescence intensity) for Bmax and 34±7.8 nM for Kd (dissociation constant).
Figure 5
Figure 5. Diffusion curves of FAM-SP after binding with different amounts of NK1R-NLPs.
Sample a to e represent 80 µL FAM-SP binding with 0.5, 4, 8, 12, 20 µL NK1R-NLP complexes respectively. Sample f to h represent 20, 10, 4, 2 µL FAM-SP binding with 10 µL NK1R-NLPs complexes respectively.
Figure 6
Figure 6. Saturation binding curve of NK1R-NLPs to FAM-SP.
[NK1R-NLPs] is the concentration of free NK1R-NLPs at equilibrium and was calculated from the subtraction of the amount of NK1R-NLPs bound with FAM-SP from the total amount of NK1R-NLPs added. The ligand bound % was calculated by comparing the average diffusion time of the mixture (free FAM-SP and FAM-SP bound with NK1R-NLPs) with the individual controls (diffusion times of free FAM-SP and free NK1R-NLPs). The experimental data (blue dots) were fitted to an “OneSiteBind” model Y  =  Bmax × X/(Kd + X). Y represents the percentage of bound ligand in the total amount of ligand, and X represents the concentration of NK1R-NLPs in the solution after reaction. The fitting results in 36±5.6 nM for Bmax and 83±33 nM for Kd.
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Grants and funding

This work was supported by funding from the University of California Discovery Grant Program, which is jointly funded by the University of California and Life Technologies Corporation. Funding was also provided by the National Science Foundation through the Center for Biophotonics Science and Technology Center, managed by the University of California, Davis under Cooperative Agreement No. PHY 0120999. Work was also performed under the auspices of the U.S. Department of Energy under contract number DE-AC52-07NA27344. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.