Crystallizing Membrane Proteins in Lipidic Mesophases. A Host Lipid Screen

Abstract

The default lipid for the bulk of the crystallogenesis studies performed to date using the cubic mesophase method is monoolein. There is no good reason however, why this 18-carbon, cis-monounsaturated monoacylglycerol should be the preferred lipid for all target membrane proteins. The latter come from an array of biomembrane types with varying properties that include hydrophobic thickness, intrinsic curvature, lateral pressure profile, lipid and protein makeup, and compositional asymmetry. Thus, it seems reasonable that screening for crystallizability based on the identity of the lipid creating the hosting mesophase would be worthwhile. For this, monoacylglycerols with differing acyl chain characteristics, such as length and olefinic bond position, must be available. A lipid synthesis and purification program is in place in the author's laboratory to serve this need. In the current study with the outer membrane sugar transporter, OprB, we demonstrate the utility of host lipid screening as a means for generating diffraction-quality crystals. Host lipid screening is likely to prove a generally useful strategy for mesophase-based crystallization of membrane proteins.

Figure 1

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 bilayer 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 bicontinuous bilayered reservoir of the cubic phase into a sheet-like or lamellar domain (A) and locally concentrate therein in a process that progresses to nucleation and crystal growth (B, from reference 9). Cocrystallization 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; β₂-adrenergic receptor-T4 lysozyme fusion; PDB code 2RH1), bilayer and aqueous channels (dark blue) have been drawn to scale. The lipid bilayer is ~40 Å thick.

Figure 2

Predicted topology map for OprB. The prediction was performed using the PRED-TMBB web server. Residues 1 to 31 correspond to the signal peptide and are not shown. Extracellular loops (L) and periplasmic turns (T) are numbered sequentially from the N- to the C-terminus. Predicted transmembrane β-strands are shown between horizontal lines representing the aqueous/apolar interfaces of the membrane. The topology suggests that the protein crosses the membrane as an 18-stranded β-barrel. The composition of the mature protein is as follows: 27 Ala, 17 Arg, 25 Asn, 36 Asp, 2 Cys, 28 Gln, 19 Glu, 47 Gly, 7 His, 12 Ile, 34 Leu, 24 Lys, 3 Met, 18 Phe, 16 Pro, 18 Ser, 19 Thr, 15 Trp, 22 Tyr, 34 Val.

Figure 3

Purification of OprB. Size exclusion chromatographic analysis of OprB (A). V_o and V_t mark the void and total column volumes, respectively. The purity of the OprB used in crystallization trials is illustrated by SDS-PAGE analysis of a loading series (B). For PAGE analysis, samples were heated at 95 °C for 5 min in Laemmli buffer before loading on to a polyacrylamide gel composed of a 12 %(w/v) resolving and a 4 %(w/v) stacking gel. The latter was removed before staining with Coomassie Blue. Purity was estimated at >90 %.

Figure 4

Micro-crystals of OprB growing at 20 °C in the lipidic cubic mesophase formed using monoolein. Small crystal are seen as dark flecks when viewed with normal light (A) and as bright flecks when viewed with polarized light (B). The contrast and brightness of the image in panel B have been adjusted to make the small crystals a little more obvious. The precipitant solution contained 25 %(v/v) tri-ethylene glycol, 0.1 M ammonium sulphate and 0.1 M glycine. Other conditions are as described in Section 2.2.3. The precipitant (P), mesophase bolus (M) and micro-crystals (X) are appropriately labelled.

Figure 5

Crystals of OprB obtained in the lipidic mesophase formed by monoolein following extensive optimization. The precipitant used in this case contained 22 %(v/v) tri-ethylene glycol, 0.1 M glycine, 0.05 M ammonium sulphate, and 0.1 M sodium acetate, pH 5.0, and the trial was conducted using 50 nL mesophase and 800 nL precipitant solution at 20 °C for 14 days. Images recorded with normal and with polarized light are shown in panels A and B, respectively. Other conditions are as described in Section 2.2.3 and in the legend to Figure 4.

Figure 6

Crystals observed growing in the lipidic cubic phase formed by 8.8 MAG. Precipitant conditions include 21 %(v/v) tri-ethylene glycol, 0.1 M ammonium sulphate, 0.1 M glycine. Micro-crystals appeared in 3 days at 20 °C. Images recorded with normal and with polarized light are shown in panels A and B, respectively. Other conditions are as described in Section 2.2.3 and in the legend to Figure 4.

Figure 7

OprB crystals growing in the cubic mesophase formed by 8.7 MAG. Precipitant conditions include 20 %(v/v) tri-ethylene glycol, 0.08 M ammonium sulphate, and 0.15 M glycine. Crystals appeared on day 3 and reached maximum size of 50 μm on day 7. Images recorded with normal and with polarized light are shown in panels A and B, respectively. Other conditions are as described in Section 2.2.3 and in the legend to Figure 4.

Figure 8

OprB crystals growing in the cubic mesophase formed by 6.9 MAG. Precipitant conditions include 21 %(v/v) tri-ethylene glycol, 0.05 M ammonium sulphate, and 0.15 M glycine. Crystals appeared after 16 h and grew to a maximum size of 40 μm × 100 μm in 7 days. Images recorded with normal and with polarized light are shown in panels A and B, respectively. Other conditions are as described in Section 2.2.3 and in the legend to Figure 4.

Figure 9

OprB crystals growing in the cubic mesophase formed by 7.8 MAG. Precipitant conditions include 26 %(v/v) tri-ethylene glycol, 0.1 M ammonium sulphate, and 0.15 M glycine. Crystals appeared after 16 h and grew to a maximum size of 20 μm × 100 – 300 μm in 7 days. Images recorded with normal and with polarized light are shown in panels A and B, respectively. Other conditions are as described in Section 2.2.3 and in the legend to Figure 4.