Protein Crystallization Hits

Membrane Protein Crystallization with Additives in LCP

The question I get asked most often when the topic comes to crystallization of membrane proteins in lipidic cubic phases (LCP) is: "what screens can I use?" (for the non-specialists: LCP stands for Lipidic Cubic Phases; these are materials that have been used successfully to serve as host matrices for the crystallization of membrane proteins). The answer is: pretty much everything. After all, the precipitants that have been used successfully are not that different from other protein crystallizations (stop reading here if you're not interested in the details).

The question regarding precipitants and additives goes way back when the LCP crystallization methodology was still in its infancy: I'm talking last century here. Due to their utility in the crystallization of hepta-helical membrane proteins, specifically GPCRs, many crystallizers are interested in using additives for LCP-mediated membrane protein crystallization. Turns out that there's a substantial body of published literature that you can take as a guide through this 'lipid swamp'. To make things simple, let's divide up these additives in lipids, detergents, small molecule amphiphiles and the more traditional crystallization reagents, such as organic solvents, polymers and salt:

Lipidic cubic phases can be doped with a variety of amphiphiles, ranging from LIPIDS such as Cholesterol, DOPE, DOPC, DMPC and PLPC to DETERGENTS (dodecyl maltoside, CTAB, beta octyl glucoside, MEGA9, DTACI and Zwittergent) (see fig 1 below).

Figure 1: Lipid and detergent additives that have been added to LCP to modulate the outcome of membrane protein crystallization experiments. These tables and the one below were taken from a chapter in this book: "14. X-Ray Crystallography of Membrane Proteins: Concepts and Applications of Lipidic Mesophases to Three-Dimensional Membrane Protein Crystallization" G protein-coupled receptors, T.Haga and G.Berstein, editors, 2000, CRC press. P. p 365-388, Loewen, M., Chiu, M., Widmer, C., Landau, E.M., Rosenbusch, J.P., Nollert.

The HOST LIPID that forms a cubic phase is likewise a factor that can be varied:

Figure 2: LCP forming lipids: monopalmitolein, monoolein, monovaccenin, monoeicosenoin and a special cyclopropyl derivative of monoolein.

Indeed, the test protein bacteriorhodopsin crystallized well in a variety of lipids and lipid mixtures:

Figure 3. Crystallization in LCP consisting of different lipids and lipid mixtures, including cholesterol.

That's a lot of compounds that can be screened: LCP (consisting of a range of lipids) with and without additives, at a variety of concentrations. At the time this wealth of possibilities felt like opening "Pandora's Box": while we realized that the LCP method is useful for many membrane protein crystallization trials, the number of possible combination of crystallization components seemed daunting. Regardless, there was a lot of excitement to apply the LCP methods to other membrane proteins, such as bovine rhodopsin or the beta-2-adrenergic receptor.

There's nothing fundamental really, that has changed since these early days. The main progress we've seen in the last 10 years was miniaturization, possibly automation, and setup formats that give better visibility to the crystals.
Foremost, however are more facile protein engineering capabilities and the fact that monoolein has worked so well as the basis host lipid that have made it so simple to apply the LCP method to any membrane protein crystallization trial.

When it comes to the more conventional crystallization reagents, all standard crystallization reagents that are used for soluble proteins can be applied. Some may not be compatible with lipidic cubic phases at all, but this property may actually cause the embedded membrane protein to crystallize (think sponge phases).

 

Figure 4. Effect of water soluble compounds on cubic phase stability. Polyethylene (PEG) and modified polyethylenes have been used to grow crystals of a variety of membrane proteins. Here is an early paper documenting this:

M. L. Chiu, P. Nollert, M. C. Loewen, H. Belrhali, E. Pebay-Peyroula, J. P. Rosenbusch and E. M. Landau
Crystallization in cubo: general applicability to membrane proteins
Acta Cryst. (2000). D56, 781-784 

Again, all conventional protein crystallization screens can be (and have been) applied to LCP-based membrane protein crystallization. Alternatively, a subset of those conditions that are compatible with the existence of LCP or with protein mobility can be screened using speciality screens, such as Emerald BioSystems' Cubic Screen.

The composition of the crystallization cocktail with all the salts, buffers and additives is described in this Cubic Screen tech-sheet.
There you'll see that alcohols such as ethanol, 2-propanol, 1,4-butanediol, PEGS (400, 1000, 3000, 8000, derivatized PEG 2000 MME), salts (sodium chloride, lithium sulfate, magnesium chloride, zinc acetate, Ammonium sulfate, sodium citrate, Sodium Malonate ) and buffers (Mes, acetate, citrate, hepes, Na/K phosphate, cacodylate, citrate, imidazole) were selected from then exisiting crystallization formulations. To get a variety of concentrations of these precipitation reagents, simple dilution of the entire screen is common practice.

Customers of the Cubic LCP kit have used these components for more than 8 years to screen crystallization conditions and grow membrane protein crystals.

All the best,
Peter

P.S. More on this topic here: Membrane Protein Crystallization In Meso: Lipid Type-Tailoring of the Cubic Phase