1UCLA-DOE Institute for Genomics and Proteomics, University of California Los Angeles, 2Department of Physiology, David Geffen School of Medicine, UCLA
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Ujwal, R., Abramson, J. High-throughput Crystallization of Membrane Proteins Using the Lipidic Bicelle Method. J. Vis. Exp. (59), e3383, doi:10.3791/3383 (2012).
Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane bilayer that surrounds all cells and organelles. Alterations in the function of MPs result in many human diseases and disorders; thus, an intricate understanding of their structures remains a critical objective for biological research. However, structure determination of MPs remains a significant challenge often stemming from their hydrophobicity.
MPs have substantial hydrophobic regions embedded within the bilayer. Detergents are frequently used to solubilize these proteins from the bilayer generating a protein-detergent micelle that can then be manipulated in a similar manner as soluble proteins. Traditionally, crystallization trials proceed using a protein-detergent mixture, but they often resist crystallization or produce crystals of poor quality. These problems arise due to the detergent′s inability to adequately mimic the bilayer resulting in poor stability and heterogeneity. In addition, the detergent shields the hydrophobic surface of the MP reducing the surface area available for crystal contacts. To circumvent these drawbacks MPs can be crystallized in lipidic media, which more closely simulates their endogenous environment, and has recently become a de novo technique for MP crystallization.
Lipidic cubic phase (LCP) is a three-dimensional lipid bilayer penetrated by an interconnected system of aqueous channels1. Although monoolein is the lipid of choice, related lipids such as monopalmitolein and monovaccenin have also been used to make LCP2. MPs are incorporated into the LCP where they diffuse in three dimensions and feed crystal nuclei. A great advantage of the LCP is that the protein remains in a more native environment, but the method has a number of technical disadvantages including high viscosity (requiring specialized apparatuses) and difficulties in crystal visualization and manipulation3,4. Because of these technical difficulties, we utilized another lipidic medium for crystallization-bicelles5,6 (Figure 1). Bicelles are lipid/amphiphile mixtures formed by blending a phosphatidylcholine lipid (DMPC) with an amphiphile (CHAPSO) or a short-chain lipid (DHPC). Within each bicelle disc, the lipid molecules generate a bilayer while the amphiphile molecules line the apolar edges providing beneficial properties of both bilayers and detergents. Importantly, below their transition temperature, protein-bicelle mixtures have a reduced viscosity and are manipulated in a similar manner as detergent-solubilized MPs, making bicelles compatible with crystallization robots.
Bicelles have been successfully used to crystallize several membrane proteins5,7-11 (Table 1). This growing collection of proteins demonstrates the versatility of bicelles for crystallizing both alpha helical and beta sheet MPs from prokaryotic and eukaryotic sources. Because of these successes and the simplicity of high-throughput implementation, bicelles should be part of every membrane protein crystallographer′s arsenal. In this video, we describe the bicelle methodology and provide a step-by-step protocol for setting up high-throughput crystallization trials of purified MPs using standard robotics.
Bicelle based crystallization is comprised of four basic steps (Figure 2): i) preparation of a bicelle forming lipid:amphiphile mixture; ii) incorporation of purified protein into the bicelle medium; iii) crystallization trials (manually or robotically); and iv) visualization, crystal extraction and freezing. These steps are described in detail below
1. Preparation of bicelles
Bicelles can form in a variety of lipid:amphiphile combinations and over a wide range of concentrations. Therefore, an initial composition-based on previous successful conditions- is recommended (Table 1). The most successful mixture is the DMPC:CHAPSO bicelle formulation, which can either be purchased commercially as a premixed ready-to-use formulation (see Table of Reagents below) or prepared in the lab as described. For this exercise we will prepare 1 ml of 35% DMPC:CHAPSO mixture at a 2.8:1 molar ratio.
2. Incorporation of protein into bicelles
Most MP structures obtained from bicelles were crystallized in DMPC:CHAPSO bicelle concentration ranging from 2 to 8% using a protein concentration of 8 to 12 mg/ml (Table 1). If possible, initial screens should use these guidelines and additional concentrations can be screened at the optimization stage. Compared to the LCP method, protein incorporation with bicelles is a simple process (Figure 3), which should be done the same day as crystallization trials.
3. Setting up crystallization trials
Other lipid crystallization techniques like the LCP require specialized equipment due to the medium′s high viscosity; but the unique phase behavior of bicelles permits implementation in virtually any standard crystallization format including robotics (Figure 3). Crystallization trials can be carried out in either hanging or sitting drop formats using standard commercially available screens.
4. Visualization, crystal extraction and freezing
Since crystal trials with the protein-bicelle mixture have a viscosity similar to protein-detergent drops, visualization and crystal extraction is routine and is carried out like traditional set-ups.
5. Representative Results:
It generally takes 2-3 days for crystals to appear and approximately a week or more for them to grow to their maximum size. This was the case for bacteriorhodopsin and mouse voltage-dependent anion channel 1 (mVDAC1) crystals4,8. For other membrane proteins it can take several weeks for crystal growth, so it is important to continue monitoring the crystal trials well beyond the first weeks.
As with other lipidic media, bicelles tend to form shapes that may appear to be crystalline. It has also been observed that they lead to a higher percentage of salt and detergent crystals. A UV-microscope that detects tryptophan fluorescence can significantly help eliminate such non-proteinaceous false positives. Figure 4 shows lipid shapes, salt and protein crystals as viewed in visible and UV light to help distinguish the different results that may be observed.
Figure 1. Bicelle Schematic. Bicelles are composed of a bilayer forming lipid molecule such as DMPC (blue) and an amphiphile such as CHAPSO (green), which protects the hydrophobic edges of the bilayer. As the temperature is increased, disc-like bicelles undergo a phase transformation into a perforated lamellar sheet12.
Figure 2. Flowchart for the bicelle crystallization method outlining the four basic steps.
Figure 3. Crystal trials set-up schematic. Purified detergent-solubilized membrane proteins can be directly mixed with bicelles on ice by simply pipetting the contents together. After incubating the protein/bicelle mixture on ice for ~30 minutes, crystallization trials can be set up using any standard format including robotics.
Figure 4. Visualization of crystal trials. Visible image (top panel) and UV image (bottom panel) of (A) Needle-shaped crystals observed in a salt-only condition. No fluorescence can be detected from the crystals, an indication of false positive. (B) Rod-shaped crystal formed in an MPD condition. The crystal fluoresced weakly but was found to be non-proteinaceous using X-ray diffraction. (C) Crystal observed about four weeks after setting up trials. The strong fluorescence under UV light confirms it is a protein crystal.
|No.||Protein||Source||Bicelle Formulation||Protein Concentration||Detergent1||Resolution (Å)||Reference|
|1||Bacteriorhodopsin2||Halobacterium salinarum||8% DMPC:CHAPSO (2.8:1)||8 mg/ml||2.0||Faham and Bowie, 2002|
|8% DTPC:CHAPSO (3:1)||8 mg/ml||1.8||Faham et al., 2005|
|2||β2-Adrenergic receptor/Fab complex||Homo sapiens||8.3% DMPC:CHAPSO (3:1)||10 mg/ml||DDM||3.4/3.7||Rasmussen et al., 2007|
|3||Voltage-dependent anion channel 1||Mus musculus||7% DMPC:CHAPSO (2.8:1)||12 mg/ml||LDAO||2.3||Ujwal et al., 2008|
|4||Xanthorhodopsin||Salinibacter ruber||4.2% DMPC, 5% NM||4 mg/ml||DDM||1.9||Luecke et al., 2009|
|5||Rhomboid protease||Escherichia coli||2% DMPC:CHAPSO (2.6:1)||9 mg/ml||Nonyl glucoside||1.7||Vinothkumar, 2011|
1 Detergent used for membrane protein purification
2 Native lipids from purple membranes may be carried along during purification
Table 1. Summary of crystallization conditions for membrane protein structures solved using bicelles.
Bicelles are a unique lipidic media that offer a native bilayer-like environment while behaving as if solubilized by detergents. This property gives bicelles a distinct advantage over other lipid-based crystallization methods since there is no learning curve or specialized equipment required for this technique. Once bicelles are available, either commercial or prepared in the lab, they can be directly mixed with purified protein and from this point on crystallization trials proceed almost exactly as with standard detergent based protocols. Furthermore, bicelles offer several practical advantages compared to other techniques, including extended storage periods, simple incorporation of protein, high-throughput capability using standard robotics and routine visualization and crystal extraction. Another distinct advantage is the ability to dope bicelles with specific lipids for optimization or if shown beneficial for the protein of interest. Taken together, the lipidic bicelle method offers considerable versatility in combination with practical ease-of-use, making it easily adoptable for all membrane protein crystallization projects.
No conflicts of interest declared.
We would like to thank Drs. James Bowie and Salem Faham for providing technical expertise and guidance on the bicelle method and Dr. Aviv Paz for useful discussions. We acknowledge Le Du for experimental support. Rachna Ujwal has financial interest in MemX Biosciences LLC, which, however, did not support this work. This work was supported in part by grants from the NIH (RO1 GM078844).
|Ready-to-use Bicelles||MemX Biosciences||MX201001/MX201002|
|Crystallization Screens||Qiagen, Hamptop Research, Molecular Dimensions, Emerald Biosystems, Jena Bioscience||Standard commercially available screens can be used for initial screening|
|Crystallization Set-up||Standard manual and/or robotic set-up available in lab can be used.|