Conformational dynamics of single G protein-coupled receptors in solution

Abstract

G protein-coupled receptors (GPCRs) comprise a large family of seven-helix transmembrane proteins which regulate cellular signaling by sensing light, ligands, and binding proteins. The GPCR activation process, however, is not a simple on-off switch; current models suggest a complex conformational landscape in which the active, signaling state includes multiple conformations with similar downstream activity. The present study probes the conformational dynamics of single β(2)-adrenergic receptors (β(2)ARs) in the solution phase by Anti-Brownian ELectrokinetic (ABEL) trapping. The ABEL trap uses fast electrokinetic feedback in a microfluidic configuration to allow direct observation of a single fluorescently labeled β(2)AR for hundreds of milliseconds to seconds. By choosing a reporter dye and labeling site sensitive to ligand binding, we observe a diversity of discrete fluorescence intensity and lifetime levels in single β(2)ARs, indicating a varying radiative lifetime and a range of discrete conformational states with dwell times of hundreds of milliseconds. We find that the binding of agonist increases the dwell times of these states, and furthermore, we observe millisecond fluctuations within states. The intensity autocorrelations of these faster fluctuations are well-described by stretched exponential functions with a stretching exponent β ~ 0.5, suggesting protein dynamics over a range of time scales.

Figure 1

Structure of β₂AR-TMR in active (orange) and inactive states (gray). Panel (a) shows a cytoplasmic view with the TMR labeling location indicated by space-filling spheres. Panel (b) shows the side view with the full agonist BI-167107 (small green structure on extracellular side) and a sketch of the surrounding DDM detergent micelle (shaded).

Figure 2

Bulk fluorescence lifetime (a, 8 ps bins) and time-resolved fluorescence polarization anisotropy decays (b, 80 ps bins) of TMR on ligand-free and agonist-bound receptors. The agonist-bound receptors display a longer fluorescence lifetime and higher anisotropy of the TMR reporter dye.

Figure 3

Examples of intensity-lifetime traces with 10 ms bin size from single trapped ligand-free receptors. Real-time intensity (blue) and fluorescence lifetime (green) are shown with calculated intensity change-point states overlaid (thick lines). Transitions among a range of intensity-lifetime states are observed, in which intensity may be correlated (b,c), uncorrelated (d), or anti-correlated (e) with lifetime. Because trace (d) shows a large, uncorrelated change in lifetime for constant intensity, we replace the thick line for mean lifetime with 1σ error bars calculated from the 10 ms binned lifetime.

Figure 4

Peak-normalized density plots of the intensity-lifetime states for the full population of ligand-free (a) and agonist-bound single receptors (b). The color bar indicates the number of states observed in each (15 cts/10 ms) × (0.25 ns) bin. Agonist-bound receptors show a tighter distribution of states with higher peak intensity and lifetime compared to ligand-free receptors. The diagonal line is a guide to the eye, highlighting states of constant radiative lifetime. In (c) and (d) we show typical lifetime decays (black) from states at coordinates ○ and +, which include the fitted lifetime component (green) together with the background component (red).

Figure 5

Histograms of state dwell times for the full population of ligand-free (a) and agonist-bound receptors (b). The mean dwell times are estimated from a single exponential fit with time constant τ_dwell. Agonist-bound receptors show longer state dwell times than ligand-free receptors.

Figure 6

Intensity autocorrelation of individual intensity-lifetime states longer than 200 ms. We show example autocorrelations (a,b,c,d) calculated with linear bins (gray), logarithmic bins (black), and we include 95% confidence bounds on zero (dashed) and the mean signal photon count rate of the state (upper right). The autocorrelation (a) is from the state in Fig. 3(a), and (b) is from the state in Fig. 3(d). We use a stretched exponential fit (green) rather than a single exponential fit (purple) because it more accurately describes the data. We show histograms of mean decay times (e) and stretching exponents (f) for ligand-free and agonist-bound receptors.