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Tips for crystallizing membrane proteins in lipidic mesophases

PSI-SGKB [doi:10.1038/th_psisgkb.2010.04]
Technical Highlight - February 2010
Short description: Advice on this useful technique for crystallizing integral membrane proteins.

One in ten integral membrane protein structures in the Protein Data Bank have been solved using a crystallization technique known as the in meso method. Recent successes with this technique include the structures of human-engineered β2-adrenergic and adenosine A2A G protein-coupled receptors.

The technique involves making an artificial lipid bilayer incorporating the protein of interest in which it can be crystallized. The bilayer is formed initially in a highly ordered cubic mesophase and is shifted by addition of 'precipitants' to a second mesophase from which the protein can crystallize. The difficulty lies in obtaining the initial cubic phase, which is difficult to work with as it is extremely viscous and sticky, almost like toothpaste in texture, making it difficult to handle and to dispense accurately and reproducibly in nanoliter volumes.

The advantage of the in meso approach is that the target protein is taken out of the potentially harmful environment of a detergent micelle (in which the protein was solubilized), and is instead placed in a more natural environment.

Caffrey and Cherezov provide a detailed description of the technique in Nature Protocols, and suggest that it would be more popular if it were easier to use. Many have tried the method but have given up.

Here, based on their protocol, we provide a few tips on how to get this technique working.

Tip 1

The phases formed by the lipid monoolein depends on temperature and hydration as illustrated in this phase diagram. The cubic (Pn3m in the figure) phase, from which membrane crystals grow at 20 °C, forms when the lipid is fully hydrated (A in the figure). Drawings of the various phases, in which the lollipops represent lipid and colored zones represent water, are arrayed around the phase diagram.

Understand what you're trying to do. The phase diagram for monoolein (see figure), the lipid most widely used to form the bilayer, shows how its phase changes with water content and temperature. At room temperature, the initial cubic phase is achieved with roughly 40% (weight for weight) water.

Tip 2

Begin using just buffer, rather than your precious protein sample, to get used to the technique and the equipment.

Tip 3

The initial cubic phase is essential, but it can be easily disrupted by the detergents, salt and solvents in the crystallization mix. Always check the phase under the conditions you intend to use. This can be done by small-angle X-ray scattering (SAXS) or by polarized light microscopy (PLM).

Tip 4

Many lipids oxidize easily so keep handling times to the minimum. Also, lipids such as monoolein are hygroscopic — they readily take up moisture — and so it is important to allow them to adjust to room temperature first.

Tip 5

To optimize crystallization conditions, standard approaches such as changing the buffer, temperature, salt concentration and adding additives are worth trying. Also, in meso optimization can be achieved by altering the composition of the lipid bilayer. So the lipid used to form the bilayer can be changed or a lipid additive can be included. Note that when using an additive, take care not to destabilize the cubic phase.

Tip 6

Various commercial kits are available, as are the individual syringe components; the authors developed their own set-up using the individual syringes. An in meso crystallization robot has also been developed along with various products to ease in meso screening.

Related articles

Membrane proteins in situ

Five good reasons to use single protein production for membrane proteins

Tips for crystallizing membrane proteins

Maria Hodges

References

  1. M. Caffrey & V. Cherezov Crystallizing membrane proteins using lipidic mesophases.
    Nature Protoc. 4, 706-731 (2009). doi:10.1038/nprot.2009.31

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