Nanoscale high-content analysis using compositional heterogeneities of single proteoliposomes

Abstract

Proteoliposome reconstitution is a standard method to stabilize purified transmembrane proteins in membranes for structural and functional assays. Here we quantified intrareconstitution heterogeneities in single proteoliposomes using fluorescence microscopy. Our results suggest that compositional heterogeneities can severely skew ensemble-average proteoliposome measurements but also enable ultraminiaturized high-content screens. We took advantage of this screening capability to map the oligomerization energy of the β2-adrenergic receptor using ∼10(9)-fold less protein than conventional assays.

Figure 1

Surface immobilization and fluorescence microscopy imaging allow for single-proteoliposome characterization. (a) Proteoliposomes tethered through a biotin-NeutrAvidin linker to a polymer-passivated (PLL-g-PEG/PLL-g-PEG-biotin) glass surface. Proteoliposomes are labeled with a lipid-coupled dye (Oregon Green DHPE) and harbor GPCRs labeled with either Cy3 or Cy5 for quantification of receptor oligomerization by FRET. (b–f) Micrographs of typical β₂-AR proteoliposome samples with nominal 1:1,000 protein-to-lipid ratio. (b) Magnified version of a typical confocal image. The assay allows high-throughput sampling of ~1,000 proteoliposomes per frame. (c–f) Micrographs and line scans showing high signal to noise for Oregon Green, Cy3, Cy5 and FRET. Gray shading highlights, respectively, an example of a proteoliposomes that carry only donor-labeled receptors (liposome i) and an example of an empty liposome (liposome iii). Exc., excitation wavelength. Color scales represent intensity in arbitrary units. Scale bars, 10 μm (b) and 1.2 μm (c–f).

Figure 2

Direct imaging of single nanoscale proteoliposomes allows for high-content analysis of intrasample compositional heterogeneities. (a) Protein aggregates, empty liposomes and proteoliposomes harboring only donor (D), only acceptor (A) and both donor and acceptor (D+A) subpopulations within a β₂-AR reconstitution sample with nominal 1:1,000 protein-to-lipid ratio (P/L). Only proteoliposomes having both β₂-AR–Cy3 and β₂-AR–Cy5 (52% ± 3%) are selected for FRET analysis. Data in the panel include N = 6,265 particles. Error bars indicate s.d. of technical replicates from 3 independent experiments. (b) Protein aggregates, empty liposomes and proteoliposome subpopulations within reconstitution samples of CB₁ and opsin receptors (both nominal P/L = 1:1,000) reconstituted by removal of detergent by either Bio-Beads (darker shades) or rapid dilution (RD; lighter shades). Data in the panel include N = 11,076 particles (CB₁), N = 11,585 (CB₁ RD), N = 10,701 (Opsin), N = 15,943 (Opsin RD). Error bars for darker bars: s.d. of technical replicates for ≥8 microscope chamber positions. Error bars for lighter bars: s.d. of technical replicates from 3 independent microscopy experiments. (c) Effect of titrating the nominal P/L (data were collected for both β₂-AR R333C and 265C; Online Methods). Data were fit to power functions to guide the eye (dashed lines). (d) Histogram of observed receptor densities for individual proteoliposomes (β₂-AR, nominal P/L = 1:1,000). (e) Histogram displaying A/D ratios on individual proteoliposomes (β₂-AR, nominal P/L = 1:1,000). All histograms include 12,809 proteoliposomes from 7 independent experiments. In d and e the red traces indicate histograms of absolute errors from single proteoliposomes.

Figure 3

Quantification of β₂-AR association energy using heterogeneities in a single reconstitution sample and ~6 pg of receptor. (a) Histogram of single proteoliposome FRET efficiencies (E_FRET) (blue) and corresponding histogram of E_FRET errors (red; histogram is shown in linear scale in Supplementary Fig. 5). The black bar marks the calculated ensemble-average E_FRET. Nominal protein-to-lipid ratio (P/L) = 1:1,000. (b) E_FRET (color scale) as a function of the total reduced receptor density (C_t) and reduced acceptor density (C_a) for a population of 921 proteoliposomes with a narrow size distribution of 120–130 nm (nominal P/L = 1:1,000). The unitless reduced densities correspond to receptor surface density multiplied by the square of the Förster radius (R₀²) (Supplementary Note). For better visualization, E_FRET values of single proteoliposomes in b,c are binned, and a weighted average of each bin is displayed. (c) Weighted fit of the data going into b (without binning) with the theoretical model (equations (11), (14) and (16) in the Supplementary Note). From the fit we extract two fitting parameters: the dimer association constant K_a and the FRET efficiency within a dimer E_bound (E_b). Bins are constructed to match those in panel b. (d,e) Residuals representing the difference between the fit in c and experimental data in b, here displayed as the percentage deviation, suggest no systematic deviations from the theory. (f) Table displaying K_a and E_b obtained by fitting the theoretical scheme (equations (11), (14) and (16)) to proteoliposomes incubated with no ligand, a saturating amount of agonist (isoproterenol (ISO), 10 μM) or an inverse agonist (ICI 118,551, 500 nM).