Growth Of 2d Crystals

2D crystals can be defined as crystals in which a motif, a protein, or assembly of proteins in the case of a protein crystal, is repeated at the points of a 2D lattice. Thus, a 2D crystal is one that is characterized by a unit cell that periodically repeats at precise locations in two dimensions but not in the third. Note that a 2D crystal is not a 2D object but is 3D. The nomenclature used to describe space groups of 2D crystals was first described by Holser (45) and revived by Fuller et al. (37). The first symbol describes the type of lattice and the second symbol the class of symmetry perpendicular to the crystal. The following symmetry operations describe symmetry elements within the plane of the crystal. There are a number of excellent reviews of membrane protein crystallization, and some of these are devoted to growth of 2D crystals (50,55,60). Thus, I will not go into great detail describing these techniques here, but will offer my own perspective.

2.1. Naturally Occurring Crystals

One of the first membrane proteins studied by electron crystallography, bacte-riorhodopsin, occurs naturally in patches of 2D crystalline "purple membrane" within the plasma membrane of Halobacterium halobium. The purple membrane can be readily purified and consists of large 2D arrays of bacteriorhodopsin molecules arranged on a 2D lattice and belonging to the 2-sided space group p3. This was one of the specimens exploited by Henderson and Unwin to demonstrate the efficacy of recording noisy electron micrographs at low electron doses and then recovering a high resolution image of a single unit cell by averaging thousands of unit cells within a crystal (78). They subsequently also calculated the first 3D image of a membrane protein and at a resolution sufficient to demonstrate transmembrane a-helices (43). A number of other membrane proteins occur naturally in 2D crystals; these include gap junctions and the photosyn-thetic membrane of the bacterium Rhodopseudomonas viridis (68,76). Others can be induced to form 2D crystals within their native membranes by the addition of specific ligands (62,71), by removing lipid enzymatically (59), or by other methods (61), and since membrane proteins are inserted the same way into their native bilayers, these crystals are all of the type shown in Figure 1a. It is also possible to form 2D crystals of a membrane protein by expressing a recombinant form in high concentration in another type of cell, as was the case with cardiac gap junction protein (76). The latter approach affords the opportunity to modify the protein using recombinant DNA technology, either by removing components that might inhibit crystallization or to identify the location of a specific sequence in the final structure.

2.2. Growth by Detergent Extraction

In some cases, it is possible to form 2D crystals of a protein by extracting native membranes in which it is present at high concentration using detergent to remove not only excess lipid but also other contaminating proteins. For example, cytochrome c oxidase constitutes approximately 10% of the protein of the mitochondrial inner membrane, and one can purify a membrane fraction of nearly pure cytochrome oxidase by treating beef heart mitochondria with appropriate concentrations of detergents followed by centrifugation to separate detergent solu-bilized components from the membrane residue (30,66,80). This requires 2 to 3 detergent treatments, and in each case, the pellet becomes enriched in cytochrome oxidase while other membrane proteins and excess phospholipid are removed by decanting the supernatants. Depending upon the type and concentration of deter gent used, two different crystal forms have been obtained. Multiple extractions with nonionic Triton® X-114 and X-100 produces a vesicular preparation of nearly pure cytochrome oxidase and 25% by weight of residual phospholipid. The molecules of cytochrome oxidase are arranged as dimers related by a crystallographic 2-fold axis. The crystal is formed when a large vesicle collapses causing molecules from two sides of the vesicle to interdigitate in the center of the vesicle, as shown in Figure 1b, forming a 2D crystal in the space group p22{21 with unit cell dimensions of a = 100 A, b = 125 A, and a thickness of 210 A (42). In this case, each unit cell of the crystal contains one molecule from each layer of the

Molecular Packing

Figure 1. Molecular packing in four classes of 2D crystal. (a) All molecules oriented in the same direction in a single bilayer. (b) One crystal composed of two layers of a collapsed vesicle. (c) Molecules with alternating orientation in a single bilayer. (d) A crystal of class in panel a rolled into a cylinder producing a structure with helical symmetry.

Figure 1. Molecular packing in four classes of 2D crystal. (a) All molecules oriented in the same direction in a single bilayer. (b) One crystal composed of two layers of a collapsed vesicle. (c) Molecules with alternating orientation in a single bilayer. (d) A crystal of class in panel a rolled into a cylinder producing a structure with helical symmetry.

collapsed vesicle, i.e., each unit cell contains two dimers. The space group describes a primitive orthorhombic lattice with a 2-fold axis of rotation perpendicular to the membrane. The symmetry operators 2j indicate 2-fold screw axes (rotation by 360°/2 followed by translation by half of a unit cell) parallel to the a and b crystal axes in the plane of the crystal.

Treatment with sodium deoxycholate removes a large proportion of phospholipid and dissociates cytochrome oxidase dimers into monomers. The resulting 2D crystal is not vesicular but consists of sheets of cytochrome oxidase monomers arranged on a primitive 2D lattice in the space group ^12j, denoting no symmetry perpendicular to the membrane and a 2i screw axis of symmetry along one of the crystal axes (37). These crystals are of the class shown in Figure 1c. The unit cell dimensions are a = 69 A and b = 140 to 170 A depending upon the preparation; the thickness is approximately 110 A (35,37). In both cases, the structures of the preparations are more complex than described here. The vesicular p22^ dimer crystals are predominantly multilayered vesicles with many small 3D crystals containing layers of the 2D crystal motif stacked in register (42,51,80). Single 2D crystals are more rare but can readily be found. The p121 crystal form contains single layer crystals as well as stacks of the simple 2D crystal motif in which successive layers are offset from one another, and electron micrographs show several differently appearing projections (35). Multilayered crystals of membrane proteins are commonly observed and often severely inhibit structural study because of the heterogeneity in the structures of different crystals (35,54,69).

2.3. Reconstitution of Purified Protein with Purified Lipids

The most general method for growing 2D crystals of membrane proteins begins with purified detergent solubilized protein and detergent solubilized lipids, either "native" lipids purified from the same type of membrane as the protein are derived or synthetic lipids using Procedure 1 (55,60,82,84). This is described in more detail in Chapter 11.

❖ Procedure 1. Growth of 2D Crystals

1. Dissolve lipids in an organic solvent (e.g., chloroform) and dry a measured amount on the surface of a suitable vessel under a stream of nitrogen.

2. Suspend the dried lipid in an appropriate volume of buffered detergent by sonication, producing mixed deter-gent—lipid micelles.

3. Mix the protein and lipid solutions approximately 1—10 mg/mL) to give a relatively high protein to lipid ratio (approximately 1:1 by weight).

4. Remove the detergent by dialysis or by adsorption.

As the detergent concentration decreases, the protein—detergent micelles and the protein—lipid micelles merge and eventually form bilayer membranes containing a high concentration of protein. Under the proper conditions, the protein molecules within the bilayers arrange themselves onto a 2D lattice forming 2D crystals (see Figure 2) (50,55,81). There are two common methods to remove detergent. One is to adsorb excess detergent by adding commercially available resin beads, e.g., Bio-Beads® (Bio-Rad Laboratories, Hercules, CA, USA) (81). A slower more controlled method is to dialyze the detergent—protein—lipid solution against detergent-free buffer. The speed of this process depends on the characteristics of the detergent employed, principally the critical micelle concentration (cmc)(50,55). In aqueous solution, detergent molecules aggregate to form micelles that are in equilibrium with individual detergent molecules; the cmc is essentially the concentration of individual molecules in the presence of micelles. Since detergent micelles are too large to pass through the pores of common dialysis tubing, dialysis of detergent solutions proceeds by the movement of individual detergent molecules through the dialysis tubing. Thus, the rate of dialysis depends on the cmc; the higher the cmc, the faster dialysis occurs. Of course dialysis also proceeds more rapidly at higher temperature. Triton detergents have low cmc's, so dialysis even at room temperature takes a relatively long period of time. For this reason, Weiss et al. used the Bio-Bead adsorption method to crystallize Complex III (cytochrome c oxi-doreductase) purified in Triton X-100 from Neurospora mitochondria (81,84). Kim et al. grew crystals of purified cytochrome c oxidase essentially identical to the dimer crystals described above by reconstitution with purified phospholipids (53).

Many factors can affect the prospects of success in crystallizing a membrane protein. As with most crystallization experiments, a critical factor is the protein sam ple which should be pure and monodisperse. The latter property can be difficult to achieve in the case of membrane proteins which, owing to their hydrophobic surfaces, can adopt multiple aggregation states even in the presence of detergents. Thus, the choice of detergent can be critical and affects both the aggregation of the protein and the methods employed in removing it during crystallization trials. For example, Suarez et al. found that beef heart mitochondrial cytochrome c oxidase is polydisperse in many common detergents, but became monodisperse when transferred to dodecyl-maltoside (also known as lauryl maltoside), a detergent consisting of a maltose polar head and a 12 carbon saturated hydrocarbon tail that they synthesized for this purpose (now commercially available) (70). Choice of detergent also affects the crystallization process, since, as mentioned above, the cmc determines the dialysis rate, and other properties affect the efficacy of adsorption to beads. Frequently, two detergents within the same generic class can have significantly different cmcs. For example, dodecyl-maltoside has

a cmc of 0.15 mM, while decyl-maltoside has a cmc of 1.6 mM. In some cases it may be desirable to slow down dialysis of a detergent with a high cmc, and this can be accomplished by dialyzing the sample against a buffer containing the detergent at a concentration that is below its cmc and then reducing the detergent concentration when the dialysate is changed. Useful information on detergent properties can be found in Jap et al. (50) and in Crystallization of Membrane Proteins edited by Michel (60) as well as in a pamphlet published by Calbiochem-Novabiochem (6).

Other factors to be considered and varied are:

• Protein concentration (generally several mg/mL).

• The presence of other solutes that might influence protein conformation (specific ligands, etc.) or solubility.

Given all of the conditions that might be varied, it is useful or essential to have a convenient system for microdialysis of many different samples. There are a number of commercial microdialysis cells suitable for this purpose. One can also construct microdialysis cells from glass tubing similar to those originally described by Zeppenzauer for crystallization of soluble proteins (87). Convenient and inexpensive microdialysis cells can also be constructed from a standard microcentrifuge tube by cutting off the conical tube leaving the tube cap and collar. In this case the compartment is formed by the tube cap over which one lays a small piece of dialysis tubing that is then sealed with the collar of the tube (Dr. Alok Mitra, personal communication).

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