Protein Crystallization Hits

This is the continuation of a target agnostic survey of often used protein crystallization reagents, based on data obtained from the Biological Macromolecule Crystallization Database (BMCD ver. 4.03). The question I'm trying to address is: which buffers and salts should you inventory?  

Covering protein crystallization space with PEGs seemed a simple affair: a set of only 12 different Polyethyleneglycols is sufficient to formulate ca. 88% of all PEG-based protein crystallization conditions.  

The situation is much less clear cut for buffers and salts that are relevant to protein crystallization. Shy of half of all protein crystallizations listed in the (BMCD ver. 4.03), 45%, can be carried out with 8 different buffers (see Fig. below). Tris buffer seems to be the champion. I interpret this as a result of investigator bias rather than there being a solid scientific reason for this buffer to play such an important role. My explanation is that neutral pH Tris buffers dominate the lab bench, and researchers take what they find first… If this is in fact true, it could support the notion that the nature of the buffer is of somewhat low importance for many protein crystallizations.

Salts are much more interesting since they  can have a dramatic effect on water properties and protein surface decoration, both affecting the ordered association of protein molecules into a crystal. Ammonium sulfate, the classic protein precipitation reagent is the clear winner. Curiously, several of the salts, such as citrates, phosphates and acetates, - the ones that provide both high ion strength and pH buffer capacity are fairly high ranked.

Popular protein crystallization buffers and salts as extracted from the Biological Macromolecule Crystallization Database (BMCD ver. 4.03)

When it comes to warehousing stock solutions for simple and quick preparation of optimization screens, these buffers: CHES, CAPS, Bicine, Tris, Hepes, Imidazole, Bis-Tris, MES

and these salts: magnesium acetate, lithium nitrate, calcium chloride, zinc acetate, potassium/sodium tartrate, sodium citrate, sodium chloride, sodium phosphate, potassium citrate, magnesium sulfate, lithium chloride, calcium acetate, ammonium phosphate, ammonium sulfate are a good start. 

Supplier, name and the associated number of stock solutions that are required for the production and optimization of protein crystallization hits. How this data was generated: Here at Emerald Bio we produce a lot of sparse matrix screens and we accomplish this with our fleet of Matrix Maker instruments that are instructed from a database of screen definitions. Since we keep track of many crystallization screens  we can identify the number of stock solutions that are used in a number of commercial protein crystallization screens.

In average there are 40 different stocks (+- 22) that are required for these protein crystallization screens. 

That's a lot of stock solutions.

Some of you may know that Emerald BioStructures, as part of the Seattle Structural Genomics Center for Infectious Disease (SSGCID) has contributed to submitting more than 444 protein structures to the PDB in the past 4 years. That's quite an achievement and my congratulations go out to the project teams that are behind these structures, most of them determined via X-ray crystallography. Some of this output, including methods used to achieve this level of productivity, are described in the  September 2011 issue of Acta Cryst F.

One of the protein production methods that has been key for several of my own 2011 protein crystallization projects: lysis scouting with the Protein Maker instrument (described in this open access article "The Protein Maker: an automated system for high-throughput parallel purification". 

Smith, E., Begley, D., Anderson, V., Raymond, A., Haffner, T., Robinson, J., Edwards, T., Duncan, N., Gerdts, C., Mixon, M., Nollert, P., Staker, B., & Stewart, L. (2011). The Protein Maker: an automated system for high-throughput parallel purification Acta Crystallographica Section F Structural Biology and Crystallization Communications, 67 (9), 1015-1021 DOI: 10.1107/S1744309111028776

What is lysis scouting?

Stated simply, lysis scouting combines the testing of a set of cell-lysis buffer conditions with IMAC (ion metal affinity chromatography) . This is done to increase the yield of proteins that appear partially soluble or insoluble under standard lysis buffer conditions.  This procedure results in a clear path forward for scaled-up production of purified protein samples for protein crystallization trials.

How is lysis scouting done?

A single batch of protein expressing E.coli cells is split into 12 pools and lysed by sonication in 12 different buffer conditions. The paper shows as an example P450 51 A1 (CYP51A1) with a 6xHis-Smt tag. This is the outline of the lysis scouting protocol:

1. Prepare 12 aliquots, each corresponding to 3 g of wet cell paste
2. Resuspend in 30 mL lysis buffer (one out of an array of 12) - see table below.

Cell lysis buffers for testing lysis conditions of recombinantly expressed fungal cytochrome P450

3. Sonicate to lyse and spin to remove cell debris

4. Clarify lysates and load on 12 x 1 mL Ni-affinity matrix column

5. Wash, elute and analyze fractions

SDS-PAGE showing that buffers 1C and 1D extract much more of the target protein CYP51A1 (red boxes). L(load), W(wash) and E(elution) fractions are shown next to MW standards.

While well expressed, CYP51A seemed insoluble using standard cell-lysis methods. The lysis-scouting procedure yielded a buffer system with a detergent (CHAPS or octyl glucoside)  in the presence of high salt concentrations (500 mM NaCl).

The utility of the Protein Maker instrument in this process is the short time it takes to run a lysis scouting experiment. Total run time is approximately 1.5 hours (excluding sample analysis). I.e. many proteins can be tested for optimal lysis conditions in a single day - and since the instrument carries out the experiment for you and in parallel, there is plenty of time to strategize the next steps of mg-scale production of the protein sample for crystallization. 

There are many protein structures that we have produced in 2011 that would not exist without Protein Maker supported lysis optimization.

A true work horse.

Peter

For the optimization of membrane protein crystal growth, it can make a huge difference if the 'proper' lipids are present in the crystallization experiment.

How do you add lipids to a crystallization trial? Lipids don't readily dissolve in water. There are several ways to get these amphipathic molecules to participate in the crystal formation process. When dealing with a standard vapor diffusion crystallization optimization, where the membrane protein detergent complex is combined with a precipitant solution, the lipid can be added in detergent micellar form. Better yet use less detergent and prepare a lipid film inside a glass container that is then solubilized by the detergent that is present in the solublized membrane protein sample. 

This makes it very simple to change the lipid composition in every single experiment.  A systematic approach to study the effect of lipids at comparably high concentrations has recently been described as HiLiDe:

Gourdon, P., Andersen, J., Hein, K., Bublitz, M., Pedersen, B., Liu, X., Yatime, L., Nyblom, M., Nielsen, T., Olesen, C., Møller, J., Nissen, P., & Morth, J. (2011). HiLiDe—Systematic Approach to Membrane Protein Crystallization in Lipid and Detergent Crystal Growth & Design, 11 (6), 2098-2106 DOI: 10.1021/cg101360d

The described re-lipidation is reminiscent of reconstitution of membrane proteins into bilayer membranes, while avoiding the second detergent removal step. I talked to the lead author Pontus earlier this year at the Keystone Conference and he explained to me that he thinks the lipid/detergent mixtures at high concentrations may form a generic crystallization environment for membrane proteins, somewhat reminiscent of lipidic cubic phases or sponge phases. The results (crystallization of  rKv1.2-beta2, E.coli Complex I and T.thermophilus Complex I and similar, previously reported crystallizations with high lipid composition such as pea LHC-II, bovine rhodopsin, bovine Cyt bc1, SERCA1a, pig Na/K-ATPase, Na/K-ATPase) speak for themselves.  

On the other hand, if the crystallization is carried out with the use of lipids that can spontaneously form a range of lipidic materials such as sponge or lipidic cubic phases, additional lipids can be added to the matrix lipid for crystal growth optimization. Let's say for instance, the matrix lipid is monoolein (good choice, by the way). This lipid has a melting temperature of 37C and in its liquid form can dissolve other membrane components, such as Cholesterol. How can this be done practically? To test three different Cholesterol concentrations, one can prepare a 20% Cholesterol in Monoolein mix by melting (i.e. 80 mg) Monoolein to 40C and dissolving the dry Cholesterol  (20 mg) in it, obtaining a 20% (w/w) mixture. Combining  this mixture with neat liquid Monoolein at a 50/50 ratio, one would obtain a 10% Cholesterol content (etc.).  Fortunately, Monoolein and lipid mixtures with Monoolein can be supercooled (i.e. remain liquid at room temperature for many minutes), allowing simple manipulation with pipettors or syringes prior to mixing with the membrane protein to form a lipidic cubic phase (or other lipidic materials).

In case you're still not convinced about the utility of amphiphilic compounds in membrane protein crystallization, a good case is made here:

NOLLERT, P. (2005). Membrane protein crystallization in amphiphile phases: practical and theoretical considerations Progress in Biophysics and Molecular Biology, 88 (3), 339-357 DOI: 10.1016/j.pbiomolbio.2004.07.006

;)

Regards,

Peter

Once you've got a hit with your membrane protein crystallization trial, your life may get really exciting. After confirming that the hit is actually a protein crystal and obtaining even the weakest X-ray diffraction patterns, you're getting into the optimization game. One of the obvious parameter to optimize is the crystallization cocktail. How can that be done without wasting your precious protein sample on unproductive crystallization conditions?  

Designing and making grid-screens around a particular hit condition is complicated due to the presence of detergent in the crystallization experiment. An established approach is to be close to the detergent phase separation boundary while avoiding the 'heartland' of phase separation. How can this be done practically? You could check out the various papers on this topic and spend a lot of time searching and possibly finding hints on formulations that are relevant to your hit. You could also search the phase boundary conditions in a pre-screen type of experiment (a la Song & Gouaux: Membrane protein crystallization: application of sparse-matrices to the alpha-hemolysin heptamer Methods in Enzymology(1997) (60-73)). The quickest way though to obtain guidance on optimization screen design is through mining of existing detergent phase boundary data. Fortunately Mary Koszelak-Rosenblum et al. from HWI have made such data mining a quick and simple experience:

Koszelak-Rosenblum M, Krol A, Mozumdar N, Wunsch K, Ferin A, Cook E, Veatch CK, Nagel R, Luft JR, Detitta GT, & Malkowski MG (2009). Determination and application of empirically derived detergent phase boundaries to effectively crystallize membrane proteins. Protein science : a publication of the Protein Society, 18 (9), 1828-39 PMID: 19554626

The data comes in an Excel spreadsheet called SLICKSPOT.  This tool is available for download here.

The input parameters for this nifty tool are type and concentrations of:

• Detergent (data is available for C10M, C12M, b-HG, b-OG, b-NG, CHAPS, LDAO, C12E8, C8E4, C8E5, FC-12),
• crowding agent PEG (data is available for: PEG400, PEG1000, PEG2000, PEG2000MME, PEG3350, PEG 4000, PEG5000ME, PEG6000, PEG8000, PEG20000)
• Salt (data is available for CaCl2, KCl, LiCl, Li2So4, MgCl2, Na2C3H2O4, NaCl, NaH2PO4, (NH4)2SO4, NH4H2PO4, (NH4)2HPO4)

So, let's say the membrane protein crystal hit was produced at room temperature with a formulation that contained 1% Octylglucoside,  Ammonium sulfate and PEG4K. For these conditions Slickspot produces the following output as a chart:

Example of a Slickspot output. How to read this customized chart: Salt and PEG concentrations below the blue curve form a single phase, those above are phase separated. Sticking to conditions around the blue line is preferred.

So, what's to do from here?

Design your customized formulation screen with Ammonium sulfate and PEG4000 conditions  near to the blue line.

Pretty slick, hm?

Peter

PS: I just realized that Slickspot fails to update the legend when additional conditions are queried; a cosmetics-only bug I hope.