Protein Crystallization Hits

Membrane protein crystallographers have become very creative in exploring materials that can serve as a matrix for membrane protein crystallization. A few years ago Salem Faham in the lab of James Bowie @ UCLA published a recipe to crystallize bacteriorhodopsin using bicelle preparations. Recently this method received some more attention in the GPCR crystallization field. As a consequence researchers are reading up on these 'old' papers to dig out the protocols for such exotic bicelle-based crystallizations.

Faham S., Bowie, J.
Bicelle Crystallization: A New Method for Crystallizing Membrane Proteins Yields a Monomeric Bacteriorhodopsin Structure
Journal of Molecular Biology, Volume 316 (1), 2002 , pp. 1-6(6)

Since I've dabbled in the 'lipid swamp' myself a little, people sometimes ask me for advice on this topic. The thing is I don't have anything new to contribute, other than having reproduced the bicelle crystallization in the lab. But I'm very happy to see that the FAQ (frequently asked questions) on bicelle based membrane protein crystallization at UCLA is still up.
A great example for openly sharing tips and important technical details that sometimes don't make it into a paper. 

Fig: how to make a bicelle sandwich? Check out the FAQ.

Thanks Salem and James for keeping the FAQ site up and running!
Peter

I have to admit, it is somewhat counterintuitive that crystallizers try to crystallize proteins by finding a milieu with low protein solubility by starting with a protein solution and then dilute it. How do you lower solubility by dilution - usually 50:50?. The trick here is of course, that the protein's solubility in the new medium (i.e. the crystallization cocktail that is used to dilute the protein with) is even lower than that in the starting solution. Hence, it seems reasonable to start with a protein buffer that allows to concentrate the protein as high as possible. One way to get to such a buffer is to enhance it for a specific target by increasing the solubility of that target protein. It turns out that this is indeed a practical way to increase the success of crystal screening, mainly due to enhanced nucleation.

Aude Izaac et al. describe a simple procedure to go about such customized buffer selection:

Izaac, A., C.A. Schall and T. C. Mueser (2006)
Assessment of a preliminary solubility screen to improve crystallization trials: uncoupling crystal condition searches
Acta Crystallographica Section D: Biological Crystallography D62, 833-842.

It works by first precipitating (ugh!) the target protein with PEG and then testing its resuspension in a variety of buffers and salts. Here's the protocol :
At first 150 ul of protein solution is precipitated by PEG 8000 (ad 190 ul 40% PEG 8000) at room temperature and spin in aliquots. Then 2 ul of 1 M salt or buffer solutions are added to 18 uL to test if the protein can be re-suspended. After centrifugation (20 ul total vol) the supernatent is tested for protein content. The higher the protein concentration in the sup, the more potent the resolubilization of the buffer for your specific target protein.
In a second step the best salt/buffer combos are determined. The salt and buffers that yielded the largest effect are combined at a standard 100 mM salt and 50 mM buffer concentration and compared to the standard chromatography buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl). Then target protein samples are diluted in the new buffer and concentrated using a 10 kDa MWCO filter. The winner is the buffer that allows the protein to concentrate to the highest level without precipitation.

Fig: What goes up must go down.

Pretty nifty,
Peter

Structure, the magazine, sent me an email the other day with "the most requested full text articles your colleagues and competitors are currently downloading from Structure.". OK - let's see what's there for us protein crystallizers:

1. Conformation Dependence of Backbone Geometry in Proteins
2. Structure of the Human Dicer-TRBP Complex by Electron Microscopy
3. The Nuclear Pore Complex Has Entered the Atomic Age • Review article
4. Discovery Through the Computational Microscope • Review article
5. Structure of PAS-Linked Histidine Kinase and the Response Regulator Complex
6. Structure of IL-33 and Its Interaction with the ST2 and IL-1RAcP Receptors-Insight into Heterotrimeric IL-1 Signaling Complexes
7. Insights into How Nucleotide-Binding Domains Power ABC Transport
8. Structural Insights on the Mycobacterium tuberculosis Proteasomal ATPase Mpa
9. Fluorescence-Detection Size-Exclusion Chromatography for Precrystallization Screening of Integral Membrane Proteins
10. Secondary Structure of Huntingtin Amino-Terminal Region
11. Structure and Signaling Mechanism of Per-ARNT-Sim Domains • Review article
12. Intrinsic Domain and Loop Dynamics Commensurate with Catalytic Turnover in an Induced-Fit Enzyme
13. Quantitative Determination of the Conformational Properties of Partially Folded and Intrinsically Disordered Proteins Using NMR Dipolar Couplings • Review article
14. Structural Plasticity of Eph Receptor A4 Facilitates Cross-Class Ephrin Signaling
15. Structural Dynamics of Light-Driven Proton Pumps
16. Subunit Architecture of Multiprotein Assemblies Determined Using Restraints from Gas-Phase Measurements
17. Toward Structural Elucidation of the @c-Secretase Complex • Review article
18. Structural Basis for DNase Activity of a Conserved Protein Implicated in CRISPR-Mediated Genome Defense
19. Structural Basis of Novel Interactions Between the Small-GTPase and GDI-like Domains in Prokaryotic FeoB Iron Transporter
20. Structures and mechanisms of glycosyl hydrolases
21. Binding of Small-Molecule Ligands to Proteins:''What You See''Is Not Always''What You Get'' • Review article
22. Structural Basis for Calcium Sensing by GCaMP2
23. Structural Basis for Binding of Hypoxia-Inducible Factor to the Oxygen-Sensing Prolyl Hydroxylases
24. The Origin of Allosteric Functional Modulation: Multiple Pre-existing Pathways • Review article
25. A Specific Cholesterol Binding Site Is Established by the 2.8 A Structure of the Human @b"2-Adrenergic Receptor

I am rather surprised by the high number of membrane protein-related papers. Out of these 25 hottest STRUCTURE articles I'd say that #9 looks like the most interesting one:

T. Kawate & E. Gouaux (2006)

Fluorescence-Detection Size-Exclusion Chromatography for Precrystallization Screening of Integral Membrane Proteins

Volume 14 (4), 673-681

And this is of course Eric Gouaux & Toshimitsu Kawate's clever paper on analyzing the monodispersity and stability of protein-detergent complexes: 

Schematic pre-crystalline screening for membrane protein crystallization.

Described is a "rapid and efficient precrystallization screening strategy in which the target protein is covalently fused to green fluorescent protein (GFP) and the resulting unpurified protein is analyzed by fluorescence-detection size-exclusion chromatography (FSEC). This strategy requires only nanogram quantities of unpurified protein and allows one to evaluate localization and expression level, the degree of monodispersity, and the approximate molecular mass." This helps leapfrogging the otherwise tedious step purification optimization. And it helps to reduce "...unproductive large-scale protein expression and purification..." efforts.

Well done!

Peter

Since I've been asked, here's a list of protein crystallization practices that may be useful to reduce your work load for protein crystallization:

Spend less time with purification - set up trials with semi-pure samples that are difficult to purify any further:
Dirty protein - is purity overrated? Just be aware that this may backfire - since there are reports of accidential protein crystallizations: AcrB and OmpF may just be the tips of the iceberg.

Skip following up false-positive crystallizations :
The single most useful practice to reduce effort in protein crystallization

Eliminate the sample concentration step: concentrate in-situ:
Avoiding the protein concentration step prior to setting up crystallization experiments

Wait longer by giving your protein crystallization experiments more time:
Protein Crystallization by dehydration

Don't work on target proteins that don't crystallize and focus on those that are easy to crystallize:
The biggest SECRET ever for success in protein crystallization

Avoid loosing precious protein samples by storing them properly:
Best Practices for storing protein samples

Happy Spring Break,
Peter