Crystallization of Membrane Proteins in Lipidic Mesophases

A typical outline of an in meso crystallization experiment is shown in Fig.1^1,2. Pre-crystallization LCP-FRAP assays are optional; however, they can significantly accelerate the process of searching for initial crystallization conditions, especially in the case of difficult membrane proteins³.

1. Protein Reconstitution in LCP

1. Purify a membrane protein of interest in a detergent solution and concentrate the protein/detergent complexes to ~10 – 20 mg/mL, taking care not to over-concentrate the detergent^1,4.
2. Transfer ~25 mg of an LCP host lipid (typically monoolein) or a lipid mixture into a 1.5 mL plastic tube and incubate at 40 °C for few minutes until the lipid melts.
3. Attach a syringe coupler to a 100 μL gas-tight syringe.
4. Load the syringe with the molten lipid using an adjustable volume pipette. Record the volume of the lipid in the syringe.
5. Load another 100 μL syringe with the protein solution at a protein solution-to-lipid ratio 2/3 v/v.
6. Connect both syringes together through the syringe coupler.
7. Push the syringe plungers alternately to move the lipid and protein through the inner needle of the coupler, back and forth, until the lipid mesophase becomes homogeneous. LCP forms spontaneously upon mechanical mixing, and the protein becomes reconstituted in the lipid bilayer of LCP. Formation of LCP can be verified by its transparent and gel-like consistency and by the absence of birefringency when viewed under a microscope equipped with cross-polarizers, or, if possible, by using small-angle X-ray diffraction¹.

2. LCP-FRAP Pre-crystallization Assays

LCP-FRAP assays are designed to measure the diffusion properties of membrane proteins reconstituted in LCP at a variety of screening conditions³. The long-range diffusion of membrane proteins in LCP is essential for successful crystallization; however, the microstructure of LCP constrains diffusion of large proteins or oligomeric protein aggregates. A common reason for failure of an in meso crystallization experiment is a fast protein aggregation leading to a loss of diffusion. It has been shown that the aggregation behavior of a protein depends on the particular protein construct, the host lipid and the composition of the screening solution³.

1. Label the protein with a fluorescent dye (Cy3 or similar) at a protein/dye ratio of ~100/1, remove the unreacted dye and concentrate the protein to ~ 1 mg/mL. Label either free amines or free thiols. When labeling free amines, use pH between 7 and 7.5 to predominately label the free N-terminus. Be aware that amino labeling can also label lipids co-purified with the protein^2,3.
2. Reconstitute the labeled protein in LCP as described in section 1).
3. Set up assay plates as described in section 3) using LCP-FRAP screening solutions instead of crystallization screens².
4. Incubate the plates at 20 °C in the dark for at least 12 hours to achieve an equilibrium state.
5. Place one of the plates on the LCP-FRAP station and focus on the first well using a 10x objective.
6. Acquire 5 fluorescent images to capture the initial pre-bleached state.
7. Trigger the laser. The laser power and number of pulses should be adjusted to bleach ~30 – 70% of the labeled protein in the middle of the bleached spot.
8. Immediately after triggering the laser, start recording a fast post-bleaching sequence of ~200 images at the fastest possible rate.
9. Follow with recording of a slow post-bleach sequence of ~50 images, selecting the delay between images as 1-20 s, depending on the diffusion rate of the protein.
10. Integrate the intensity inside the bleach spot in all frames and correct it for bleaching and light intensity fluctuations during the acquisition by dividing the intensity inside the bleached spot by the averaged intensity of a reference spot outside of the laser bleached area.
11. Normalize the corrected intensity to make the pre-bleached intensity equal to 1 and the initial bleached intensity equal to 0.
12. Fit the curve of the normalized intensity vs. time, F(t), using the following equation⁵:
    F(t) = M x exp(-2T/t) x (I₀(2T/t) + I₁(2T/t)), (Eq.1)
    where M is the mobile fraction of diffusing molecules, T is the characteristic diffusion time, t is the real time of each recorded frame, I₀ and I₁ are the 0^th and 1^st order modified Bessel functions.
13. Calculate the diffusion coefficient, D, as:
    D = R²/4T, (Eq.2)
    where R is the radius of the bleached spot.
14. Move to the next well and repeat steps 2.5) – 2.13).
15. Compare the mobile fractions and diffusion coefficients obtained for the different screening conditions. Design new crystallization screens based on the components that facilitated protein diffusion and excluding conditions for which protein diffusion was not observed. If the protein did not diffuse in any of the screened conditions, consider broadening the screening space or trying a new protein construct.

3. Setting Up LCP Crystallization Trials

1. Reconstitute the protein in LCP as described in section 1).
2. Transfer the protein-laden LCP into a 10 μL gas-tight syringe attached to a repetitive syringe dispenser.
3. Attach a short removable needle (gauge 26, 10 mm length) to the 10 μL syringe.
4. Dispense 200 nL boluses of LCP on the surface of four adjacent wells forming a 2×2 square.
5. Overlay each of the LCP boluses with 1 μL of corresponding crystallization screen solution.
6. Cap four loaded wells with an 18 mm square glass coverslip. Apply a gentle pressure on the coverslip to seal the wells.
7. Repeat steps 3.4)-3.6) with the next set of 4 wells until the whole plate is filled.
8. Incubate the plate at a constant temperature, periodically checking for crystal formation and growth.

4. Harvesting Crystals from LCP

1. Place a plate with protein crystals under a stereo microscope with variable zoom, equipped with a linear rotating polarizer and analyzer.
2. Focus on the well of interest using a low power zoom so that the whole well is placed within the field of view.
3. Score the coverslip glass in four strokes making a square inside the well boundaries using a sharp corner of a ceramic capillary cutting stone.
4. Press around the scored perimeter with strong sharp-point tweezers to propagate the scratches through the thickness of the coverslip glass.
5. Punch two small holes at opposite corners of the scored square.
6. Inject few μL of precipitant solution through one of the holes to reduce dehydration during the subsequent steps.
7. Using an angled sharp needle probe break up the glass along one or two sides to free the cut-out square.
8. Carefully lift up the glass square watching for the cubic phase bolus. If the bolus is stuck to the coverslip, then flip the glass square over and place on the bottom of the well.
9. Add an extra few μL of precipitant solution, supplemented with a cryo-protectant, if necessary, on top of the exposed cubic phase bolus in the well.
10. Increase magnification of the microscope and focus on a crystal.
11. Adjust the angle between the polarizer and the analyzer to increase the contrast between the birefringent crystal and the background, while keeping enough light to see the harvesting loop.
12. Select a MiTeGen MicroMount with a diameter matching the crystal size and then harvest the crystal directly from the LCP by scooping it into the MicroMount.
13. Flash freeze the MicroMount with the harvested crystal in liquid nitrogen, and ship it to a synchrotron source beamline for X-ray data collection⁶.

5. Representative Results:

An engineered human beta 2 adrenergic G protein-coupled receptor (β₂AR-T4L) was expressed in baculovirus infected sf9 insect cells and purified in dodecylmaltoside (DDM)/ cholesteryl hemisuccinate (CHS) detergent solution bound to a partial inverse agonist carazolol⁷. The protein was labeled with Cy3 NHS ester and used in LCP-FRAP pre-crystallization assays (Figure 2). Coarse grid screens based on several conditions selected from the results of LCP-FRAP assays produced initial crystal-like hits (Figure 3). Further optimization of precipitant conditions yielded diffraction quality crystals (Figure 4).

Figure 1. Flow-chart of a typical LCP crystallization experiment. Steps in the gray boxes are not described in the current protocols.

Figure 2. LCP-FRAP assay with β₂AR-T4L/carazolol in monoolein based LCP. A) Results of an LCP-FRAP assay performed in an automatic high-throughput mode, in which each sample of a 96-well plate is bleached sequentially and fluorescence recovery is measured after a 30 min incubation. The obtained fluorescence recoveries, which represent the mobile fraction in each sample, are plotted for all 96 samples. The screening solutions contain 0.1 M Tris pH 8, 30 % v/v PEG 400 combined with 48 different salts at two different concentrations. B) Fluorescence recovery profiles for several representative conditions. Solid line curves represent fits by Eq. 1.The mobile fractions and the diffusion coefficients are determined using Eqs. 1 and 2. Fast recovery of less than 10% in the sample containing Na chloride is due to fluorescently labeled lipids co-purified with the protein.

Figure 3. Initial crystal hits of β₂AR-T4L/carazolol obtained by a coarse grid screening around most promising conditions identified by LCP-FRAP, containing Na sulfate (panel A) and Na Formate (panel B). The protein is labeled with Cy3 NHS ester and the fluorescent images are taken using excitation at 543 nm and emission at 605 nm.

Figure 4. Optimized crystals of β₂AR-T4L/carazolol. The images of crystals grown in the presence of Na sulfate (panels A and B) and K Formate (panels C and D) are taken in the brightfield mode (panels A and C) and using cross-polarizers (panels B and D).