LCP-FRAP Assay for Pre-Screening Membrane Proteins for in Meso Crystallization

Abstract

Fluorescence recovery after photobleaching was used to study the diffusion of two integral membrane proteins, bacteriorhodopsin and beta2-adrenergic receptor, in lipidic cubic phase (LCP). We found that the diffusion properties within the LCP matrix strongly depend on the protein construct and applied screening conditions. Common precipitants often induce restriction on diffusion of proteins in LCP and thereby impede their chances for crystallization. A high protein mobile fraction and a fast diffusion rate correlate very well with known crystallization conditions. Using this knowledge, one can now pre-screen precipitant conditions with microgram quantities of material to rule out conditions that are not conducive to diffusion, nucleation, and crystal growth. The results of this assay will narrow membrane protein crystallization space by identifying suitable protein constructs, stabilizing compounds and precipitant conditions amenable to in meso crystallization. Crystallization pre-screening will significantly increase the chances of obtaining initial crystal hits, expediting efforts in generating high-resolution structures of challenging membrane protein targets.

Figure 1

Cartoon of the in meso crystallization process. Membrane protein (blue, β₂AR-T4L, PDB ID 2RH1) is embedded in the lipid bilayer of the lipidic cubic phase (low left corner). Upon addition of a precipitant the crystal nucleates. The lipidic cubic phase transforms into a multilamellar phase in the vicinity of the growing crystal and serves as a portal connecting the bulk cubic phase to the growing crystal (upper right corner). Membrane proteins are expected to diffuse in 3-dimensions within the single lipid bilayer and approach the crystal through the lamellar phase portal. If proteins form micro-aggregates (such as shown in the middle-bottom part of the cubic phase),their diffusion through narrow channels in LCP will be highly restricted and no crystals will grow.

Figure 2

FRAP measurements of the diffusion of Rhodamine 6G in 90 %w/w glycerol solution. (a) Fluorescence images recorded during the FRAP acquisition sequence. Time 0 s corresponds to the bleaching event. Images are cropped to 120 × 120 pixels area. (b) Fractional fluorescence recovery curve fitted with a one-component diffusion recovery equation. (c) Radially averaged profile of the bleached spot modeled with a Gaussian.

Figure 3

(a) Fluorescence recovery curves for bacteriorhodopsin in LCP at different concentrations of Na/K phosphate pH 5.6. Recovery curve at 50 mM salt is fitted by a one-component equation (Eq.2), while data at both 1 and 2.5 M salt required using two-component equation (Eq. 3). (b) Example of one-component versus two-component curve fitting for bR in at 1 M Na/K phosphate pH 5.6

Figure 4

(a) Dependence of the fraction of fast diffusing bR molecules on salt concentration at different pH. (b) Change in the total mobile fraction of bR in LCP over time at different concentrations of Na/K phosphate pH 5.6.

Figure 5

(a) Fluorescence recovery curves recorded for β₂AR(E122W) and β₂AR(E122W)-T4L in LCP incubated with Bis tris propane pH 7.0 and with the crystallization conditions for β₂AR(E122W)-T4L (0.1 M Bis tris propane pH 7.0, 25 %v/v PEG 400, 0.1 M Na sulfate, 5 %v/v 1,4-butanediol). (b) Two component fitting of the fluorescence recovery curve obtained for β₂AR(E122W)-T4L in 0.1 M Bis tris propane pH 7.0, 15 %v/v PEG 400, 0.1 M Na sulfate, 5 %v/v 1,4-butanediol conditions. The fast component describes diffusion of labeled lipids and the slow component – diffusion of protein.

Figure 6

Trends for diffusion coefficient and mobile fraction of β₂AR(E122W)-T4L obtained by systematically varying concentration of PEG 400 (a), Na sulfate (b) and pH (c), starting from 0.1 M Bis tris propane pH 7.0, 25 %v/v PEG 400, 0.1 M Na sulfate, 5 %v/v 1,4-butanediol. (d) Time-course dependence of the β₂AR(E122W)-T4L mobile fraction in 0.1 M Bis tris propane pH 7.0, 25 %v/v PEG 400, 0.2 M Na sulfate, 5 %v/v 1,4-butanediol (close circles) and in 0.1 Bis tris propane pH 8.0, 25 %v/v PEG 400, 0.1 M Na sulfate, 5 %v/v 1,4-butanediol (open circles). The shaded areas corresponds to concentrations of PEG 400 (a), Na sulfate (b) and pH (c) supporting β₂AR(E122W)-T4L crystallization and to the time frame (d), during which the crystals continue to grow. No diffusion was detected in (c) at pH 4.6 and the diffusion coefficient was assigned an arbitrary low value of 10⁻⁴ µm²/s to represent a point on the log scale. All measurements were made at least in duplicates. The error bars are shown for data points measured for three or more times.