Improved in Vitro Folding of the Y2 G Protein-Coupled Receptor into Bicelles

Abstract

Prerequisite for structural studies on G protein-coupled receptors is the preparation of highly concentrated, stable, and biologically active receptor samples in milligram amounts of protein. Here, we present an improved protocol for Escherichia coli expression, functional refolding, and reconstitution into bicelles of the human neuropeptide Y receptor type 2 (Y₂R) for solution and solid-state NMR experiments. The isotopically labeled receptor is expressed in inclusion bodies and purified using SDS. We studied the details of an improved preparation protocol including the in vitro folding of the receptor, e.g., the native disulfide bridge formation, the exchange of the denaturating detergent SDS, and the functional reconstitution into bicelle environments of varying size. Full pharmacological functionality of the Y₂R preparation was shown by a ligand affinity of 4 nM and G-protein activation. Further, simple NMR experiments are used to test sample quality in high micromolar concentration.

Keywords: GPCR; NMR; NPY; bicelles; folding.

Figure 1

Scheme of the three-step folding protocol for the preparation of the Y₂R in either isotropic or non-isotropic bicelles. The main steps are the folding dialysis (step 1), the reconstitution into bicelles (step 2), and concentrating the sample for NMR measurements (step 3).

Figure 2

Negative staining electron microscopy images of (A) small isotropic bicelles (q = 0.25), (B) intermediate sized bicelles, and (C) non-isotropic bicelles (q > 10). The inset in (A) shows the same sample after 1 week of storage at room temperature. Stacking of the bicelles becomes visible, which leads to reduced binding yields and substantial line broadening in solution NMR spectra. Samples from (A) and (C) are used for solution and solid-state MAS NMR, respectively. Image (B) illustrates the fusion of the small bicelles to larger patches during removal of the DHPC-c7 and hence to an increased q-value.

Figure 3

Results of the CPM assay for testing disulfide bridge formation. High fluorescence intensities designate free cysteine residues. The dotted line indicates the background fluorescence intensity. Disulfide bridges are formed during the folding process to completeness after step 3. Glutathione (GSH) accelerates the formation, which is fully reversible shown by reducing the cysteines using DTT.

Figure 4

Pharmacological characterization of the Y₂R preparation at nanomolar concentration using a fluorescence polarization assay with [Dpr²²-atto520]-NPY. The saturation assay in (A) recorded at increasing concentration of bicelle-reconstituted Y₂R (black) displays two inflection points at 4 and 126 nM. The latter represent the binding of NPY to the membrane, as revealed by the reference measurement with empty bicelles (gray). The specificity of the high affinity Y₂R binding of 4 nM is confirmed in the competition assay in (B) using increasing concentration of unlabeled NPY. The error bars were determined from three independent preparations.

Figure 5

In vitro folded Y₂R variants functionally activate purified G_i protein. (A) Y₂R folded into isotropic bicelles and activated with NPY drastically accelerates nucleotide exchange of wild type Gα_i1. The fluorescence trace is given as mean of seven independent experiments. (B) Resulting apparent rates of GTPγS binding of Gα_i1 (basal) and Y₂R-catalyzed nucleotide exchange. Statistical significance was determined using one-way ANOVA/Dunnett's post hoc test against basal Gα_i1 in Graph Pad Prism 5.03. ^###p < 0.001; ^##p < 0.01.

Figure 6

NPY binding tests of the Y₂R preparation in concentration of 50 μM using solution NMR spectroscopy. In (A) a ¹H-¹⁵N HSQC spectra of specifically labeled NPY in the presence of Y₂R with a NPY/Y₂R ratio of 2 (black) and 18 (gray) are shown. Chemical shift perturbations (CSP) were measured for the labeled NPY positions Y20, I28, Q34 which are involved in Y₂R binding (Kaiser et al., 2015), but not for A14 which is not interacting with the Y₂R. As control the same amounts of NPY were titrated to empty bicelles to exclude self-aggregation effects of NPY at high concentration. Concentration dependent binding effects were verified in (B) by observing the CSP at increasing concentrations of NPY binding to Y₂R in isotropic bicelles, and in (C) by measuring the signal intensities of bound NPY to Y₂R in non-isotropic bicelles. All spectra were recorded at 293 K.

Figure 7

Solid-state MAS NMR spectra of uniformly labeled Y₂R in non-isotropic bicelles showing ¹³C/¹³C correlation using DARR. The mixing time was varied from 20 ms (left) to 500 ms (right). In the bottom right half of the 20 ms DARR spectrum are superimposed one bond correlations cross-signals, simulated from an Y2R homology model. The measurements were performed at a MAS frequency of 7 kHz and a temperature of 5°C.