by Peter Nollert
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.
Pretty nifty,
Peter