The structural biology of membrane proteins remains a challenging fi eld, partly due to the dif fi culty in obtaining high-quality crystals. We developed the bicelle method as a tool to aid with the production of membrane protein crystals. Bicelles are bilayer discs that are formed by a mixture of a detergent and a lipid. They combine the ease of use of detergents with the bene fi ts of a lipidic medium. Bicelles maintain membrane proteins in a bilayer milieu, which is more similar to their native environment than detergent micelles. At the same time, bicelles are liquid at certain temperatures and they can be integrated into standard crystallization techniques without the need for specialized equipment.
Despite many heroic efforts and some success stories, the number of membrane protein structures determined continues to lag far behind soluble proteins. One major hurdle is the dif fi culty in obtaining good-quality crystals. For many membrane proteins, detergents are the partner that they “cannot live with, or without.” Detergents are necessary for the extraction and solubilization of membrane proteins, while at the same time they can interfere with crystal growth. By their nature, detergents cover much of the hydrophobic surface of a membrane protein leaving little surface area for crystal growth. In the absence of a sizable soluble domain, the protein contacts required for crystal growth are limited to mostly loop regions. This often leads to poor crystal quality since loops are typically the more fl exible portion of the protein ( 1 ) . This challenging task of growing well-ordered membrane protein crystals has prompted the development of a number of technical
1. Introduction
1.1. Crystallization of Membrane Proteins
4 S. Agah and S. Faham
advances. These developments include the antibody method ( 2 ) , the protein fusion method ( 3, 4 ) , and the lipidic cubic phase (LCP) method ( 5 ) . These methods have proven useful, even critical in certain cases. Nonetheless, a universal solution appears out of reach as the unique features of each protein can considerably in fl uence the crystallization process. The distinctive features of each protein may favor a certain method over others; thus the development and advancement of various approaches are bene fi cial. The success of the LCP method demonstrated that membrane proteins can be crystallized from lipid media ( 5 ) , not just from detergent media. The lipid cubic phase forms a network of interconnected bilayers that is generated by the lipid monoolein (1-cis-9-octadecenoyl)-rac-glycerol. Due to inherent challenges of the LCP method, such as its high viscosity, we explored the utility of a different lipid phase, namely, bicelles, for the crystallization of membrane proteins, and developed the bicelle method described here.
Bicelles are bilayer micelles that are formed by speci fi c mixtures of a lipid and a detergent (Fig. 1 ). Bicelles were originally used in solid-state NMR studies and continue to receive much attention in the NMR fi eld, since they tend to partially align in a magnetic fi eld ( 6 ) . We were successful in crystallizing bacteriorhodopsin in bicelles ( 7 ) , and determined its structure from two different bicelle-forming
1.2. Bicelles
Fig. 1. Representation of the morphology of bicelles. Bicelles are bilayer discs. The bilayer is formed by the lipid (DMPC), with the detergent (CHAPSO) covering the edges of the hydrophobic bilayer.
51 Crystallization of Membrane Proteins in Bicelles
compositions, demonstrating that bicelles can be a useful medium for the crystallization of membrane proteins ( 8 ) . Since our initial report, additional membrane proteins have been crystallized from bicelles con fi rming that this method holds great promise. These are the structures of β 2-adrenergic receptor ( β 2AR) ( 9 ) , voltage-dependent anion channel (VDAC) ( 10 ) , xanthorhodopsin ( 11 ) , and rhomboid protease ( 12 ) .
Bicelles are made from a mixture of 1,2-dimyristoyl- sn -glycero-3-phosphocholine (DMPC) and 1,2-dihexanoyl- sn -glycero-3-phospho choline (DHPC) or a mixture of DMPC and 3-((3-cholamidopropyl)dimethylammonio)-2-hydroxy-1-pro-panesulfonate (CHAPSO) ( 13, 14 ) . The long-chain lipid (DMPC) forms the bilayer portion of the bicelles, and the detergent mole-cules (DHPC, or CHAPSO) cover the edges (Fig. 1 ). Bicelles can form under a broad range of concentrations and lipid-to-detergent ratios. For DMPC/DHPC mixtures these ranges include (~3% to ~40%) in lipid concentration and (~1:2 to ~5:1) in lipid-to-deter-gent ratio ( 15, 16 ) . Bicelles have been examined by NMR ( 17 ) , electron microscopy (EM) ( 18 ) , and small angle neutron scattering (SANS) ( 19 ) . A signi fi cant aspect of the bicelle mixture is that its (temperature–concentration) phase diagram is complex involving a number of phase transitions ( 19 ) . By plotting the concentration on the x -axis and temperature on the y -axis it is possible to represent where each of the phases occur and where the phase transitions happen. It is important to note that bicelles form in speci fi c regions of the phase diagram. These additional phases include the perfo-rated lamellar phase, and the multi-lamellar vesicles phase (Fig. 2 ). The fi ne details of the phase diagram are a matter of debate ( 20 ) , and additional phases have been proposed such as the branched fl at cylindrical micellar phase ( 16 ) .
Phase transitions can occur as a result of variations in a number of parameters such as temperature, bicelle concentration, and bicelle composition ( 15 ) . A signi fi cant aspect of the bicelle phase is that it is a liquid, thus easy to manipulate. The lamellar phase is a gel, and may be bene fi cial for crystal growth in some circumstances. The transition from a liquid to a gel occurs as temperature is raised. A possible explanation for this seemingly unusual behavior is that as the temperature is raised more of the detergent molecules are incorporated into the bilayer and fewer detergent molecules remain available to cover the edges; as a result the bilayer portion expands and at a certain point a phase transition occurs and the gel phase forms ( 20– 22 ) . The transition temperature is in fl uenced by the bicelle composition. For example, DMPC:DHPC bicelles are a liq-uid at room temperature and form a gel over ~30°C. In contrast, bicelles prepared with 1,2-ditridecanoyl- sn -glycero-3-phosphocholine (DTPC) instead of DMPC have a lower transition temperature. DTPC:DHPC bicelles are liquid at ~12°C and form a gel at ~20°C ( 17 ) . The morphology of bicelles can also vary. The diameter of
6 S. Agah and S. Faham
bicelles has been reported to vary from 8 to 50 nm ( 23, 24 ) . The size of the bicelles is in fl uenced by the bicelle composition and the presence of divalent cations such as calcium or magnesium ( 25 ) .
Membrane protein crystals grown in lipidic media including LCP and bicelles appear to have a similar type of packing that is distinct from the packing observed for crystals grown in deter-gents. Instead of having crystal contacts limited to loop regions, substantial crystal contacts are formed by the transmembrane hydrophobic region of the proteins. It was suggested early on that membrane proteins can produce two types of crystals based on their packing ( 26 ) . In type I crystals two-dimensional layers stack on top of each other to produce a three-dimensional lattice. In each 2D layer the membrane proteins pack side by side positioned roughly as they would be in a bilayer, namely, with the planes that de fi ne the thickness of the transmembrane regions arranged in a parallel orientation (Fig. 3a ). With this type of packing, substantial crystal contacts are formed by the transmembrane region of the protein. Crystals grown in lipid media mainly produce type I crystal packing ( 27, 28 ) . In type II packing the majority of crystal con-tacts are formed by the water-exposed portion of the membrane protein, and it is mainly observed for membrane protein crystals grown from detergents ( 26 ) (Fig. 3b ).
The phase diagrams of detergents, LCP, and bicelles can be in fl uenced by the various precipitants commonly used in crystallization trials. At the same time each phase can signi fi cantly affect the
Fig. 2. The bicelle phase diagram. Temperature–concentration phase diagram for 3.2:1 DMPC:DHPC mixture, with total lipid concentration on the x -axis, and temperature on the y -axis. Although the actual phase diagram may be even more complex, this diagram pro-vides a visual illustration on the effects of temperature and concentration on the bicelle phase. Temperature alone can in fl uence the shift from the liquid bicelle phase to the gel lamellar phase. MLV represents multilamellar vesicles. This fi gure is adapted from ( 19 ).
71 Crystallization of Membrane Proteins in Bicelles
crystallization process. It has been reported for detergents that some regions in the phase diagram are more favorable to crystal growth ( 29 ) . For the LCP method, much attention has been paid to identifying compatible crystallization conditions that are able to maintain the LCP phase ( 30 ) . However, it is recognized that crystal growth in LCP may require the formation of a lamellar phase ( 31 ) . Careful analysis of the monoolein phase diagram has led to the lipidic sponge phase (LSP) method ( 32 ) , which also relies on being in a speci fi c region of the phase diagram. As for the bicelle method, results suggest that crystallization can occur in both the liquid phase, and in the gel phase. The process of crystal nucleation and growth may require unidenti fi ed local phase changes to accommodate the packing of protein molecules and to make room for growing protein crystals.
Fig. 3. Crystal packing for membrane proteins. ( a ) Type I crystal packing, protein molecules pack side by side forming a two-dimensional (2D) layer. The 2D layers align to form a three-dimensional (3D) crystal. Crystals grown from lipidic media mainly belong to type I. ( b ) Type II crystal packing. Most of the crystal contacts are formed by the soluble and loop regions. This is mainly observed for crystals grown in detergents.
8 S. Agah and S. Faham
1. Lipids: DMPC and/or DTPC (Avanti Polar Lipids, Inc.). 2. Detergents: DHPC and/or CHAPSO (Sigma-Aldrich). 3. Sonicator (Heat systems ultrasonics w385). 4. Vortex (VWR). 5. Puri fi ed protein of interest at ~10 mg/ml. The choice of deter-
gent is protein dependent (see Note 1). 6. A crystallization setup. Such 24 well trays (Hampton Research)
for manual crystallization or a high throughput robot (mos-quito from TTP labtech) for 96-well format. Both conven-tional hanging drops and sitting drops are suitable (see Note 2).
7. Lids or crystal clear tape for 96 well trays, and cover slides for 24 well trays (Hampton Research).
8. Standard commercially available crystallization screens (Hampton Research, or Qiagen).
9. A UV microscope that detects tryptophan fl uorescence (Korima Inc., PRS1000) ( 33 ) .
10. Cryoloops and tools for freezing crystals (Hampton Research).
Protein crystallization in bicelles can be described in four steps. First, the bicelle medium is prepared. Second, the protein is incor-porated into the bicelles. Third, the crystallization trials are set up. Fourth, crystallization trials are monitored and crystals extracted.
Bicelles can be prepared from a number of ingredients and at different concentrations (~3–40%) (see Notes 3 and 4).
Here is an example for preparation of a 35% bicelle solution at 2.8:1 (DMPC:CHAPSO) (see Notes 5 and 6). The 2.8:1 ratio refers to the molar ratio; it is not a direct weight:weight ratio. The 35% is a weight/volume ratio. The weight referred to in the 35% case is the sum of the weights of (DMPC + CHAPSO). For this calculation we approximate the density of the DMPC/CHAPSO mixture to be close to ~1 mg/ml ( 34 ) . Naturally, the density will change with temperature and as the composition transitions from a liquid phase to a gel phase. Therefore, to prepare 100 ml of a 35% bicelle solution the sum of the weights of DMPC and CHAPSO would be 35 g. The addition of 65 ml H 2 O results in a fi nal volume that is approximately the desired 100 ml. We fi nd this approxima-tion to be useful since the volumes we work with are much smaller than 100 ml.
2. Materials
3. Methods
3.1. Preparation of the Bicelle Mixture Stock Solution
91 Crystallization of Membrane Proteins in Bicelles
The molecular weights used for this calculation are DMPC = 677.933 g/mol and CHAPSO = 630.877 g/mol. The molecular weight of a 2.8:1 bicelle is 1,898.2124 + 630.877 = 2,529.0894. In order to prepare 0.5 ml (35%, 2.8:1) bicelles, 325 μ l water is added to 175 mg bicelles. The amount of DMPC needed equals (175 × 1,898.2124)/2,529.0894 = 131.347 mg, and the amount of CHAPSO needed is (175 × 630.877)/2,529.0894 = 43.653 mg.
1. Add 325 μ l water to 43.653 mg CHAPSO in a 1.5 ml Eppendorf tube.
2. Vortex until CHAPSO dissolves. 3. Weigh out 131.347 mg DMPC. 4. Add the dissolved CHAPSO to the DMPC. 5. Vortex. The fi nal volume should be close to 0.5 ml. 6. Sonicate the mixture in short (1 s) bursts using a microtip
sonicator. Sonication should be brief as to avoid froth formation. One to two bursts should be suf fi cient. Some recommend against son-ication, in which case extended periods of vortexing will be required. Flash freezing with liquid nitrogen may help as well.
7. Go through cycles of cooling (4°C, or on ice), vortexing, and heating (~37°C) until the sample is homogeneous (for exam-ple, ~5 cycles and 1–2 min/cycle). An indication that the bicelle phase has formed is that the mixture will be a clear liquid when kept cool, and will form a clear gel when warmed up to 37°C. It is possible to transition between these two phases several times (see Note 7).
8. Keep bicelle solution cool (~4°C) for immediate use (see Note 8).
Addition of the protein to the bicelle mixture will bring along the detergent used in the puri fi cation. Detergents will likely in fl uence the bicelle phase diagram. To minimize the effect of the detergent we try to maintain a low detergent concentration relative to the bicelle concentration. Proteins puri fi ed in DDM and LDAO have both been successfully crystallized in bicelles (see Notes 9 and 10). It is possible to purify membrane proteins from native membranes without the addition of detergents, and to crystallize successfully with the bicelle method as was the case with bacteriorhodopsin and xanthorhodopsin ( 7, 11 ) .
1. Thaw the bicelle mixture if frozen, and keep cool (~4°C). 2. You may need to vortex the mixture to reform a homogeneous
bicelle mixture, as precipitation might occur during freezing, thawing, or even as a result of prolonged periods on ice.
3.2. Preparation of the Protein/Bicelle Mixture
10 S. Agah and S. Faham
Bicelles prepared at lower concentrations (<35%), or at higher detergent-to-lipid ratio will not experience this precipitation as often, and may be easier and faster to prepare.
3. Add the protein of interest (~10 mg/ml) in a 4:1 protein:bicelle ratio while kept cold. If the initial bicelle concentration is 35%, then it will be diluted to 7%, which has been used successfully in crystallization. If the initial protein concentration is 10 mg/ml, it will only be diluted to 8 mg/ml (see Note 11).
4. Mix by pipetting up and down to homogeneity. 5. Incubate the protein/bicelle mixture on ice for 30 min with
occasional mixing to allow for complete incorporation of the protein into the bicelles. NMR can be used to assess the incorporation of the membrane protein into bicelles ( 35, 36 ) ; however we do not experimentally validate the incorporation of the protein into the bicelles.
6. If protein activity assays are available, testing the activity of the protein sample after incorporation into bicelles can be very useful to con fi rm that the protein remains active and properly folded.
One of the attractions and bene fi ts of the bicelle method is that it is possible to carry out crystallization trials using standard tools and protocols. Nonetheless, it is sensible to check the liquidity of the protein/bicelle mixture by visual inspection during the pipetting procedure as bicelles can get more viscous at room tem-perature. In case gelling is observed, then extra caution should be taken by keeping the sample cool enough to be in the liquid phase. This should not be a problem in most cases; however if the bicelle composition is altered to one that forms a gel at low temperature (for example as a result of using DTPC instead of DMPC), then additional care may be required. Also setup of the crystallization trials in a warm room should be avoided.
1. Crystallization trials can be set up by standard methods such as pipetting the protein by hand or by a nanoliter dispensing robot (see Note 12).
2. Commercially available screens are convenient to use with bicelles. However, some of the conditions included in the usual screens may lead to phase separation. For example, we have observed phase separation in condition G6 of the classics screen from Qiagen (0.1 M NaCl, 0.1 M Bicine pH 9.0, 20% PEG550MME) (see Note 13). The appearance of nonprotein crystalline material is also a concern and care must be taken when analyzing the results of the crystallization trials. Given the limited number of structures solved from bicelles, along with the fact that bicelles can be prepared from different components, it is dif fi cult to develop a complete crystallization
3.3. Crystallization of the Protein/Bicelle Mixture
111 Crystallization of Membrane Proteins in Bicelles
screen speci fi c for the bicelle method. Nonetheless, lessons can be learned from the currently available information. Protein crystals have been formed under various conditions. Salt-based screens (phosphates, and sulfates) are the most successful so far, but crystals have been obtained in polyethylene glycols (PEG2K) ( 8 ) , and organics (MPD) as well ( 10 ) .
3. After the crystal trays have been prepared, the trays can be transferred to the temperature of interest (37°C, room tem-perature, or anywhere in between) (see Note 14). Protein crys-tallization in bicelles at 4°C has not yet been reported; it is plausible, although possibly tricky as it may lead to increased precipitation of the lipids (see Note 15).
The crystallization temperature can play an important role in the outcome, as temperature can strongly in fl uence the phase behavior of the bicelle mixture. Small temperature changes of only 1–2° have been suspected to cause dif fi culty in reproducing crystal-lization results.
Identi fi cation of protein crystals in bicelles is not trivial as it may be dif fi cult to distinguish between lipid features and protein crystals. To minimize confusion we recommend one of the two approaches:
(a) Set up double drops for each condition, one with bicelles alone, and one with protein and bicelles.
(b) Use a UV microscope ( 33 ) . By relying on tryptophan fl uorescence a UV microscope can help distinguish between protein crystals and nonprotein features (Fig. 4 ) (see Note 16).
3.4. Visualization and Extraction of Crystals Grown in Bicelles
Fig. 4. UV microscope. Protein crystals can be easier to identify with the assistance of a UV microscope. Tryptophan fl uorescence can help distinguish protein from nonprotein features as long as other fl uorescent compounds are not included in the crystallization experiment.
12 S. Agah and S. Faham
Crystal extraction from bicelle drops should be no different from standard methods. The gel phase that forms with the bicelle mixture is only mildly viscous and does not prevent handling the crystals or mounting them with crystal loops. Addition of a cryo solution can help loosen the crystals from the bicelle gel if necessary.
It is possible to screen for cryo conditions using standard approaches, without a need to remove the bicelles. The bicelle mixture may provide some additional cryo protection for the crystals. Cryo conditions that have been used with the bicelle method are listed in Table 1 .
1. Detergents carried along with the protein can in fl uence the bicelle behavior. If one desires to maintain a bicelle mixture as close to the original components as possible, one needs to be careful not to carry along too much detergent with the protein. Therefore, care must be taken when the protein sample is concentrated.
2. Standard crystallization setups used for typical protein crystal-lization experiments are suitable with the bicelle method. In contrast to the LCP method, no additional specialized tools are required.
3. Successful results have been reported from a number of bicelle-forming mixtures including DMPC:CHAPSO, DTPC:CHAPSO, and DMPC:nonyl-maltoside (NM). Protein crystals have also been observed in DMPC:DHPC bicelles. Additionally, bicelles can be prepared from other lipids or detergents. Including the
4. Notes
Table 1 List of cryo conditions used for freezing protein crystals grown with the bicelle method
131 Crystallization of Membrane Proteins in Bicelles
use of 1,2-dilauroyl- sn -glycero-3-phosphocholine (DLPC) or 1,2-dipalmitoyl- sn -glycero-3-phosphocholine (DPPC) instead of DMPC ( 37 ) or the use of 3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonate (CHAPS) instead of CHAPSO ( 38 ) .
4. Lipid additives can be used in the process of screening for crys-tallization conditions. These lipids can be added at the fi rst step, the bicelle preparation stage, or the second step, where the protein is incorporated into bicelles.
5. Protein stability analysis on opsin, the apo form of rhodopsin, in bicelles showed that the protein is more stable in DMPC:CHAPS bicelles than in DMPC:DHPC bicelles. This was demonstrated by the increased resistance of the protein to urea-induced denaturation ( 38 ) . One possible explanation suggested is the increased rigidity of the DMPC:CHAPS bicelles.
6. Small amounts of hexadecyl(cetyl)trimethylammonium bro-mide (CTAB) or tetradecyltrimethylammonium bromide (TTAB) have been reported to stabilize bicelles ( 39, 40 ) . It has been also reported that DMPC:DHPC bicelles can be stabilized by the addition of cholesterol ( 41 ) or cholesterol sulfate ( 42 ) .
7. If the sample gets dispersed and stuck on the inner walls of the tube during the preparation process, centrifugation of the tube is recommended. This will maintain the proper concentration and ensure reproducibility.
8. For long-term storage bicelles can be kept at −20°C. When thawing the bicelle mixture, caution is required to ensure that the solution is homogeneous before use. Potentially, cycles of heating, cooling, and vortexing may be required.
9. The β 2AR structure showed that bicelles can be used in con-junction with the bound Fab fragment method ( 9 ) .
10. Both β -barrels and α -helical membrane proteins have been crystallized with the bicelle method ( 7, 9– 12 ) .
11. Preparing samples at a high initial bicelle concentration reduces the extent to which the protein is diluted. However, if one is able to concentrate the protein sample to high levels (~20 mg/ml), then it is not necessary to have the bicelles at a high initial con-centration. Additionally, some proteins may crystallize at lower concentrations. Xanthorhodopsin crystallized in bicelles at ~4 mg/ml concentration.
12. It is possible to chill the platform of the nanoliter dispending robot (such as the mosquito) with ice prior to applying the protein sample. Only the plate position where the protein is loaded needs to be chilled. In case the bicelle composition
14 S. Agah and S. Faham
being used has a low gelling temperature (for example DTPC:CHAPSO), then chilling may help avoid sample gell-ing. The tray setup procedure is not long, and the cooler platform can help keep the sample in the liquid phase.
13. Many of the crystallization conditions may dramatically alter the bicelle phase diagram. We do not consider these condi-tions to be incompatible with the bicelle method. On the other hand, conditions that cause lipid precipitation are incompatible.
14. Phosphatidylcholine lipids that are used to form the bicelles may slowly hydrolyze at room temperature. This process appar-ently did not interfere with the growth of bacteriorhodopsin crystals even at low pH and over extended periods of time. However, it should be noted that phosphatidylcholine lipids with an ether linkage instead of an ester linkage are able to form bicelles and are more resistant to hydrolysis ( 43, 44 ) .
15. Setting up crystallization trials at 4°C can lead to precipitation of the lipids in some of the typical crystallization conditions that have high precipitant concentrations.
16. The observation of fl uorescent crystals in the UV microscope does not guarantee that they are protein crystals. The protein may precipitate on a salt crystal and cover it completely.
Acknowledgments
The authors would like to thank Professors Jochen Zimmer and Michael Wiener for critical review of the manuscript.
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