Membrane Structural and Functional Biology Group, Schools of Biochemistry and Immunology and Medicine, Trinity College Dublin
Caffrey, M., Porter, C. Crystallizing Membrane Proteins for Structure Determination using Lipidic Mesophases. J. Vis. Exp. (45), e1712, doi:10.3791/1712 (2010).
A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the lipidic cubic phase or in meso method. The method has been shown to be quite versatile 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. Recent successes using in meso crystallization are the human engineered beta2-adrenergic and adenosine A2a G protein-coupled receptors. Protocols are presented for reconstituting the membrane protein into the monoolein-based mesophase, and for setting up crystallizations in the manual mode. Additional steps in the overall process, such as crystal harvesting, are to be addressed in future video articles. The time required to prepare the protein-loaded mesophase and to set up a crystallization plate manually is about one hour.
An important focus in the area of structural and functional biology is the biological membrane (Figure 1). The membrane, which surrounds the cell and sub-cellular organelles when present, is a molecularly thin structure just two lipid molecules across and is studded with proteins. Structure and function as applied to both lipid and proteins are of interest. However, the focus of this article is restricted to membrane proteins.
A better understanding of how membrane proteins function at a molecular level is sought for two reasons. Firstly, there is the intellectual satisfaction in knowing how they work. Secondly, by knowing how a protein works, there is always the prospect of being able to fix it should it malfunction or of improving or even modifying it for particular applications. Drug design is an obvious outcome of this type of work. One approach to figuring how a membrane protein works at a molecular level is to determine its structure. This involves establishing the location in 3-dimensional space of all atoms, or at least all non-hydrogen atoms, that make up the protein. The method we use for this purpose is macromolecular X-ray crystallography (MX). Figure 2 shows an example of a membrane protein whose structure was determined using MX. A diffraction quality crystal of the protein is required to do MX.
Clearly, there are many steps involved in structure determination using macromolecular crystallography. This is illustrated in Figure 3. Typically, these include identifying a membrane protein target, and then producing, purifying and crystallizing it. Diffraction measurements are performed on the crystal using a home or a synchrotron X-ray source. The diffraction data are processed yielding an electron density map that is then fitted with a molecular model. The model, when refined, can be used to explore the mechanism of action of the protein and for structure-based drug design.
The focus of this article is to show how we produce diffraction quality crystals of membrane proteins using lipidic mesophases, by the so-called in meso method. A recent review of the method and its scope is available in Reference 1 (Caffrey, 2009). The step-by-step protocol we will follow here is described in Reference 2 (Caffrey and Cherezov, 2009).
A flowchart summarizing the steps involved in and time required for setting up an in meso membrane protein crystallization trial is shown in Figure 4. This article covers those steps enclosed by dashed red lines.
Part 1: Preparing the Crystallization Plates
Part 2: Preparing the Lipid Syringe
Part 3: Preparing the Protein Syringe
Part 4: Mixing Protein Solution and Lipid: Making the Mesophase
Part 5: Loading the Dispenser
Part 6: Setting up Crystallization Plates
Part 7: Representative Results
The appearance of the resulting crystals will vary with the inherent color of the membrane protein, the polarization of the light used for inspection (or lack thereof) and the method and quality of illumination. Figure 6 shows several possible crystal appearances. Naturally colored membrane proteins growing in meso when viewed with normal light can look like those shown in Figure 6 (Panels b and d). Colorless membrane protein crystals growing in the cubic phase when viewed with normal light can look like those shown in Figure 6 (Panel e). Finally, colorless membrane protein crystals growing in meso when viewed with polarized light can look like Figure 6 (Panels a and c).
The next steps in the overall process of structure determination are to harvest and cryo-cool the crystals and to record and analyze X-ray diffraction from them. These topics are to be covered in separate JoVE articles.
Figure 1. Schematic representation of a biological membrane showing the lipid bilayer in and on which are situated a variety of proteins.
Figure 2. The structure of the vitamin B12 transporting protein, BtuB, solved using MX and crystals grown by the in meso method6 illustrated in this JoVE article.
Figure 3. The structure-function cycle illustrates many of the steps involved in obtaining and utilizing complete structural information about a protein.
Figure 4. The flowchart summarizes the steps involved in the production of membrane protein crystals by the in meso method. Only those steps surrounded by the dashed red line are covered in this JoVE article. From Reference 2.
Figure 5. A simplified temperature-composition phase diagram for the lipid (monoolein)/water system. Crystallization trials are set up at 20 °C where the lipid saturates with water at 40 % hydration. The detailed phase diagram is available in Reference 5.
Figure 6. Crystals of membrane proteins growing in the lipidic mesophase.
(a) vitamin B12 transporting protein, BtuB6, (b) light-harvesting complex II7, (c) the adhesin/invasin OpcA8, (d) bacteriorhodopsin9, (e) a carbohydrate transporter from Pseudomonas. Images recorded with normal light (b,d,e) and between crossed polarizers (a,c).
The cubic phase is a delicate and dynamic environment that can change drastically with the alteration of a number of variables. It is not possible to give a description of the setup of in meso crystallization trials in the manual mode that describes all potential pitfalls. However, many difficulties can be avoided by practicing the technique before applying it to expensive protein solutions and by using moderation in pressure applied to syringes during mixing. Done correctly, the in meso method can yield crystals of a wide variety of proteins, the number of which is constantly increasing.
The description given here of the setup of in meso crystallization trials is focused on the manual mode. The process can be, and often is modified to facilitate automated setup of the crystallization plates in those cases that require large-scale screening of crystallization conditions.
There are many who contributed to this work and most are from the Caffrey Membrane Structural and Functional Biology Group, both past and present members. To all we extend our warmest thanks and appreciation. This work was supported in part by grants from Science Foundation Ireland (07/IN.1/B1836), the National Institutes of Health (GM75915), and the University of Limerick.
|Purified water||Reagent||EMD Millipore|
|Disposable pipet tips||Disposable||Pipetman||Various|
|Gas-tight syringes||Tool||Hamilton Co||80265 (25-Ál)|
|Syringe tips||Tool||Hamilton Co||7770-020 (gauge 22)|
|Narrow bore coupler*||Tool||Hamilton Co||various/EBS-LCP-2|
|Repeating dispenser||Tool||Hamilton Co||83700|
|Silanized glass microscope slides||Disposable||Gold Seal||3010|
|microscope coverslips||Disposable||Fisher Scientific||12-548C|
|Perforated double-stick spacer tape||Disposable||Saunders Corporation (hole-punched)||NA|
|Brayer (roller)||Tool||Fisher Scientific||50820937|
|Chipped ice||Temperature Control||N/A||NA|
* Full details for machining your own Narrow Bore Coupler are provided in Reference 3.