April 27, 2010 17:00
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!
April 20, 2010 15:00
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.
April 6, 2010 14:00
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,