Crystallizing membrane proteins using lipidic mesophases

Abstract

A detailed protocol for crystallizing membrane proteins that makes use of lipidic mesophases is described. This has variously been referred to as the lipid cubic phase or in meso method. The method has been shown to be quite general in that it has been used to solve X-ray crystallographic structures of prokaryotic and eukaryotic proteins, proteins that are monomeric, homo- and hetero-multimeric, chromophore-containing and chromophore-free, and alpha-helical and beta-barrel proteins. Its most recent successes are the human-engineered beta(2)-adrenergic and adenosine A(2A) G protein-coupled receptors. Protocols are provided for preparing and characterizing the lipidic mesophase, for reconstituting the protein into the monoolein-based mesophase, for functional assay of the protein in the mesophase and for setting up crystallizations in manual mode. Methods for harvesting microcrystals are also described. The time required to prepare the protein-loaded mesophase and to set up a crystallization plate manually is about 1 h.

Figure 1

Temperature-composition phase diagram of the monoolein/water system determined under “conditions of use” in the heating and cooling directions from 20 °C. Redrawn from ref. . A cartoon representation of the various phase states is included in which colored zones represent water. The 20 °C isotherm is shown as a blue, horizontal line and points along it referred to in the text are labeled A – E. The liquid crystalline phases below ~17 °C are metastable. Ia3d and Pn3m represent cubic phases.

Figure 2

Cartoon representation of the events proposed to take place during the crystallization of an integral membrane protein from the lipidic cubic mesophase. The process begins with the protein reconstituted into the curved bilayers of the ‘bicontinuous’ cubic phase (tan). Added ‘precipitants’ shift the equilibrium away from stability in the cubic membrane. This leads to phase separation wherein protein molecules diffuse from the continuous bilayered reservoir of the cubic phase by way of a sheet-like or lamellar portal to lock into the lattice of the advancing crystal face (mid-section of figure). Co-crystallization of the protein with native lipid (cholesterol) is shown in this illustration. As much as possible, the dimensions of the lipid (tan oval with tail), detergent (pink oval with tail), cholesterol (purple), protein (blue and green; β2AR-T4L; PDB code 2RH1), bilayer and aqueous channels (dark blue) have been drawn to scale. The lipid bilayer is approximately 40 Å thick.

Figure 3

The in meso crystallization robot. The robot has two dispensing arms. Arm 1 includes four or eight tips and is used for handling liquid precipitant solutions. Arm 2 supports a microsyringe for dispensing the protein/lipid dispersion. The microsyringe dispenses mesophase by the action of a microsyringe pump which in turn is driven by a controller. When not in use, arm 2 goes to a park position where the microsyringe needle tip is placed in a moist sponge. Also shown in the figure are a 96-well glass plate, a 96-deep-well block of precipitant solutions and a station where the tips on arm 1 are washed.

Figure 4

A flow chart summarizing the steps involved in and time required for setting up an in meso membrane protein crystallization trial. Boxed items represent starting and intermediate materials and products. Rounded boxes represent processes identified by step number/s referred to in the text. Times are provided for the preparation of a single 27-well sandwich plate unless otherwise noted. Asterisks mark pause sites. 2x indicates that the procedure should be repeated twice.

Figure 5

The glass sandwich crystallization plate. (A) A perspective drawing of the plate consisting of a glass slide base, a perforated spacer and a glass cover slip. (B) A photograph of a 27-well plate viewed from above where each well is loaded with 1 μL of a precipitant solution and 50 nL of cubic phase containing bacteriorhodopsin. The plate has three cover slips in place. Adapted from ref. .

Figure 6

The lipid mixing device. The coupled syringe mixer is shown (A) disassembled and assembled (B, C). In (B) the mixer is shown loaded and ready for mixing with lipid (white) in one syringe and protein solution (pink) in the other. The homogenized, protein-loaded mesophase (light pink) in the syringe on the right is shown in (C).

Figure 7

Ways to set up in meso crystallization trials using commercial plates. The type of plates used includes microbatch (A), sitting drop (B), and hanging drop (C–E). In D and E a sandwich is made of the mesophase (red) by placing a small glass coverslip (hatched) below (D) or above (E) the bolus. The precipitant solution is colored a shaded pale blue, the vacuum grease is purple, and the sealing tape is gray.

Figure 8

Microsyringe repeating dispenser. (A) Side view. (B) View from plunger end.

Figure 9

Crystals of membrane proteins growing in the lipidic mesophase. A, bacteriorhodopsin; B, light harvesting complex II; C, the adhesin/invasin OpcA; D, the vitamin B12 transporter, BtuB; E, the engineered human β2 adrenergic recepton-T4 lysozyme chimera; F, a carbohydrate transporter from Pseudomonas. All crystals were obtained by the authors.

Figure 10

Images of birefringent solid and liquid crystalline lipidic phases recorded using polarized light microscopy. A. Dried out lipid mesophase. This represents a typical outcome when the precipitant solution was either not dispensed or it dried out due to incomplete sealing. It likely includes the Lc and/or L_α phases. B. Crystalline L_c phase. C. L_α phase. D. Swollen L_α phase. E. Hexagonal phase. F. Typical texture observed with a bolus incubated at pH > 9 that may have undergone hydrolysis and fatty acid production.

Figure 11

Non-birefringent ‘objects’ with sharp edges and corners that appear in lipid mesophases under in meso crystallization conditions. A and C are images observed with normal light. The corresponding images recorded between cross polarizers are shown in B and D. The angular objects likely include domains of cubic phase, voids and trapped bubbles or droplets. Arrowed objects are for cross-referencing between images recorded with and without polarized light.

Figure 12

Distinguishing protein from salt crystals. UV-fluorescence microscopy is shown here distinguishing protein (A, B) from salt (C, D) crystals growing in in meso glass sandwich plates. The protein is the engineered human β₂ adrenergic receptor-T4 lysozyme chimera and the salt is sodium sulfate. Images in A and C were recorded with normal light. Images in B and D were recorded using a Korima fluorescence microscope with excitation and emission wavelengths of 280 nm (bandpass 20 nm) and 360 nm (bandpass 40 nm), respectively. Images were recorded with plates oriented ‘upside down’ such that the glass baseplate faces the incident exciting light source and the detector. Fluorescent protein crystals and precipitate can be seen in B. The salt crystal is not fluorescent and is not visible in D.

Figure 13

A key for scoring the outcome of in meso crystallization trials. The scale runs from 0 to 9 and the corresponding images recorded in normal light (left panel) and between crossed polarizers (right panel) are shown. 0 – Birefringent mesophase. Certain precipitant solutions can trigger a conversion from the cubic to a lamellar or hexagonal phase, both of which are highly birefringent under cross-polarizers. Such mesophases are unlikely to support growth of protein crystals. 1 – Dissolved lipid. Some precipitants, such as MPD, PPO, Jeffamine M-600 and other organics, at high concentrations can dissolve the lipidic mesophase. This can often lead to the protein itself precipitating. 2 – Clear cubic phase. The cubic phase can be identified readily as being non-birefringent, transparent, and gel-like with rough edges. 3 – Precipitate. Here the protein forms a brownish, non-birefringent precipitate. This often happens in the swollen cubic phase, the sponge phase or when the lipid completely dissolves. Drops with heavy precipitate are unlikely to yield crystals. Light precipitate can indicate that the condition is close to crystal nucleation. Often crystals nucleate and grow in a drop with light precipitate. 4 – Birefringent precipitate. The protein precipitate has some birefringency under cross-polarizers. Optimization of this condition is recommended. 5 – Crystallites or spherulites. Protein forms birefringent particles that lack angularity or a well-defined crystal shape. This result can serve as a lead for optimization. 6 – Microcrystals. Crystals smaller than a few microns. Usually extensively nucleated producing “showers” of birefingent dots most apparent when viewed under cross-polarizers. 7 – Needles. Crystals grow preferentially in 1 dimension. Size in the other two dimensions is at or below the resolving power of the microscope. 8 – 2D Plates. Crystals grow in two dimensions. Plate thickness is below the resolving power of the microscope. 9 – 3D Crystal. Crystals grow in all three dimensions and all three dimensions of the crystal are clearly seen with a light microscope.

Figure 14

Small-angle X-ray diffraction patterns of hydrated monoolein in the cubic and sponge phases. Samples were prepared with water (a), apo-BtuB protein solution (b), and apo-BtuB protein solution followed by incubation with MPD-containing precipitant for 16 days at 20 °C (c). The cubic-Pn3m phase with lattice parameters of 104.8 A and 104.3 A was recorded in (a) and (b), respectively. The diffuse ring surrounding the beam stop shadow in (c) is characteristic of the L3 or sponge phase. Samples were prepared with 60 %(w/w) monoolein and 40 %(w/w) water in (a), 60 %(w/w) monoolein and 40 %(w/w) protein solution (12.9 mg BtuB/mL in 20 mM Tris/HCl pH 8.0, 0.1 M NaCl, 0.1 %(w/v) LDAO) in (b) and (c). In (c) 2 μL of the lipid/protein mesophase in an X-ray capillary tube was overlain with 10 μL of precipitant solution containing 10 %(v/v) MPD, 0.2 M ammonium formate, 50 mM MES pH 6.5 and incubated for 16 days at 20 °C before data collection. Micro-crystals of BtuB were present in (c) at the end of the incubation period. Reproduced from ref. .

Figure 15

X-ray diffraction patterns of the various phases that can occur in the monoolein/screen solution/water system. Phase identity follow: (A) Lc phase. (B) L_α phase. (C) cubic-Im3m phase. (D) cubic-Ia3d phase. (E) cubic-Pn3m phase. (F) H_II phase. (G) L_α and cubic-Ia3d phase coexistence. The cubic phase pattern is spotty because of the presence in the sample of relatively large monodomains (see ref. 46). (H) L_α phase in conjunction with extensive diffuse scattering. (I) L_α phase and disordered cubic (inner diffuse ring) phase coexistence. Diffraction images are from ref. . Bragg reflection indices are detailed in ref. .

Figure 16

Spectrophotometry/fluorescence cuvette and accessories. A. 3 mm Pathlength cuvette containing optically clear lipidic cubic phase following centrifugation. B. Teflon cuvette holder for centrifugation. The holder fits inside a 15 mL Falcon tube. C. Adapter for 3 mm cuvette that fits into a standard (1 cm × 1 cm) spectrophotometer or fluorimeter cuvette holder. D. The same adapter as in C resting on a Teflon shim to position the cuvette in the light beam of a Beckman spectrophotometer.

Figure 17

Spectrophotometric and visual properties of BtuB in detergent solution and in the cubic phase. (a) CD spectra of apo-BtuB in detergent solution and in the cubic phase. The region of the cubic phase spectrum below ~208 nm is not reliable because of strong background absorption by the lipid, as described. (b) Quenching of apo-BtuB intrinsic fluorescence by bromo-MAG in the cubic phase of hydrated monoolein. Fluorescence intensity (F_c) was normalized to the value recorded in the absence of quenching lipid (F₀). Values reported are the average of at least triplicate sample preparations. (c) Scatchard analysis for the binding of CNCbl to apo-BtuB in micellar solution (solid circles) and in the cubic phase of hydrated monoolein (open circles). The corresponding dissociation constant, Kd, values are 1.02 and 1.24 nM. (d) Photograph of a bolus of cubic phase with (i, iii) and without (ii) reconstituted apo-BtuB equilibrated for 6 days at 20 °C with a solution of 67 μM CNCbl. In (iii), the bathing CNCbl solution was replaced with CNCbl-free buffer just before the photograph was taken to make the labeling of the bolus more obvious. The bolus of cubic phase can be seen as an elliptically shaped object at the bottom of a cuvette. Adapted from ref. .

Figure 18

Initial BtuB microcrystal hits. Pictures were taken using brightfield illumination (a) and using cross-polarizers (b) on the fourth day after setup. Precipitant: 0.05 M magnesium chloride, 30 %(v/v) PEG 550 MME, 0.1 M Hepes, pH 7.5.

Figure 19

Absorption spectrum of LH2 in detergent solution and in the lipidic cubic phase. Spectra were recorded 15 min after protein dilution into detergent solution (dashed line; 0.035 mg/mL) or reconstitution in the cubic phase (solid line; 50 %(w/w) monoolein, 0.07 mg/mL). Reproduced from ref. .

Figure 20

Initial β₂AR-T4L microcrystal hits. Pictures were taken using brightfield illumination (a) and using cross-polarizers (b) on the third day after setup. Precipitant: 0.1 M lithium sulfate, 0.1 M sodium chloride, 30 %(v/v) PEG 400, 0.1 M Hepes pH 7.5.