Resolving distance variations by single-molecule FRET and EPR spectroscopy using rotamer libraries

Abstract

Förster resonance energy transfer (FRET) and electron paramagnetic resonance (EPR) spectroscopy are complementary techniques for quantifying distances in the nanometer range. Both approaches are commonly employed for probing the conformations and conformational changes of biological macromolecules based on site-directed fluorescent or paramagnetic labeling. FRET can be applied in solution at ambient temperature and thus provides direct access to dynamics, especially if used at the single-molecule level, whereas EPR requires immobilization or work at cryogenic temperatures but provides data that can be more reliably used to extract distance distributions. However, a combined analysis of the complementary data from the two techniques has been complicated by the lack of a common modeling framework. Here, we demonstrate a systematic analysis approach based on rotamer libraries for both FRET and EPR labels to predict distance distributions between two labels from a structural model. Dynamics of the fluorophores within these distance distributions are taken into account by diffusional averaging, which improves the agreement with experiment. Benchmarking this methodology with a series of surface-exposed pairs of sites in a structured protein domain reveals that the lowest resolved distance differences can be as small as ∼0.25 nm for both techniques, with quantitative agreement between experimental and simulated transfer efficiencies within a range of ±0.045. Rotamer library analysis thus establishes a coherent way of treating experimental data from EPR and FRET and provides a basis for integrative structural modeling, including studies of conformational distributions and dynamics of biological macromolecules using both techniques.

Figure 1

Cysteine positions for fluorescence and spin labeling. (A) PTBP1-RRM3/4 (blue ribbons) with labeling positions highlighted (C_α atoms as colored spheres, connected by gray arrows for S392C). Positions are offset by single turns along the two helices α₁ and α₂ (gray cylinders), resulting in a shift to smaller distance (black arrow) from E468C (blue) to S475C (green), or from Q388C (magenta) to S392C (orange) (green arrow). The flexible N-terminus is indicated by a dashed line. (B) Fluorescence- and spin-labeled cysteine side chains (stick representation) are drawn to scale with respect to (A). To see this figure in color, go online.

Figure 2

EPR distance determination by DEER and simulations. (A) Experimental DEER distance distributions (Fig. S2 shows primary data) between RRM3/4 cysteine positions as indicated in the legend, labeled with maleimido proxyl. (B) Rotamer library simulations for maleimido proxyl for all 20 conformations in the NMR ensemble (PDB: 2ADC (74)). Arrows (in A and B) indicate the shift to smaller distances from E468C (blue) to S475C (green). (C) NMR ensemble of RRM3/4 (blue ribbons, PDB: 2ADC (74)) with point clouds indicating the conformational distributions of maleimido proxyl-labeled side chains from the RLA simulations (colored spheres indicate label positions, size indicates population). Distances between the point clouds are distributed as shown in (B). To see this figure in color, go online.

Figure 3

Single-molecule FRET measurements and comparison with simulations. (A) Experimental single-molecule FRET efficiency histograms for RRM3/4 represented by the Gaussian fits of the peaks (solid lines; cf. Fig. S3 for the original histograms, which are omitted here to improve visualization of the small differences). The labeling positions are indicated (see legend), with darker or lighter colors for helix α₂ labeled with the acceptor or the donor dye, respectively. (B) Interdye distance distributions obtained from RLA simulations for all 20 conformations of the NMR ensemble (PDB: 2ADC (74)) with the same color code as in (A). Arrows in (A) and (B) indicate the shift to smaller distances or higher FRET efficiencies from E468C (blue) to S475C (green). (C) NMR ensemble of RRM3/4 (blue ribbons, PDB: 2ADC (74)) with spatial distributions of the chromophore centers (point clouds) from rotamer library simulations for fluorescence label Cy3b on Q388C and S392C and for CF660R on E468C, V472C, and S475C for all 20 conformations in the NMR ensemble. To see this figure in color, go online.

Figure 4

Comparison of experimental smFRET efficiencies to predictions using diffusional averaging over distance distributions. (A and C) RMSD between experimental and simulated FRET efficiencies averaged over all 12 data sets (see Table S3) for different Förster radii (R₀ ± 7% (86)) and effective diffusion constants of the fluorescence labels based on the distance distributions (see Figs. 3 and S12) of the RLA simulations (A) and AV simulations (C) using the same color scale given in (A). The red crosses mark R₀ = 6.0 nm and D = 0.2 nm²/ns and correspond to the circles in (B). (B) Experimental versus simulated FRET efficiencies from RLA (green) and AV analysis (black) with shaded bands corresponding to uncertainties in Förster radius R₀ and effective diffusion constant D (see A). Linear fits to the data with slope 1 (green and black lines for RLA and AV, respectively) show deviations from the ideal 1:1 correlation (gray dashed line). To see this figure in color, go online.

Figure 5

Resolving distances and distance variations by EPR and single-molecule FRET (smFRET) using RLA simulations. (A) Comparison of experimental FRET efficiencies (filled symbols, error bars represent standard deviations estimated from multiple measurements (Table S3)) with simulated results using RLA distance distributions and diffusional averaging (open symbols) for positions 388 and 392 on helix α₂ labeled with Cy3b with the second label at positions 468, 472, and 475 in blue, red, or green, respectively. (B) is analogous to (A) but for CF660R attached to α₂. (C) Comparison of DEER and RLA distance distributions oriented upwards or downwards, respectively. Labeling positions along α₂ are indicated, and positions 468, 472, and 475 on α₁ are shown in blue, red, or green, respectively. The symbols indicate the average distances. (D) Correlation between RLA average distances calculated for EPR and FRET (see Tables S1–S3 for data). (E) Labeling positions on RRM3/4 with arrows, which indicate distance variations between positions along α₁ (black) or α₂ (green) in all panels. To see this figure in color, go online.