I am working on bacterial protein, which will be crystallized for structural study. My protein MW is 22kDa and pI is 9.2. I have tried crystallization trial in 20mM Tris, 150mM KCl and 5 % glycerol. I want to know how buffer pH, conc. and salt conc. affect the crystallization.
Should I keep the same buffer in new crystallization or not. If not, then what composition modulation can be done?
First of all, make sure your protein is happy in the buffer and pH that you have. For the rest it is usually a good idea to minimise the content of the buffer, thus minimise buffer, salt and glycerol (but still make sure the protein is happy). By doing this you increase the available "crystallisation space" you have from the standard crystallisation screens. The modulation is the performed mostly by the crystallisation screens, and not but what you have along with your protein at the start.
High salt concentration increases solubility, thus you want to go as low as possible in the protein. The buffer used can play a role, but sticking with like 10 mM Tris, Hepes or similar is usually ok.
NaCl is a chaotrope (I'm assuming KCl should be too) and it will disrupt crystallisation at high concentration as it interrupts hydrogen bond networks. Therefore, trying to keep the NaCl concentration as low as possible will help crystallisation. Few proteins will be soluble and stable in the absence of NaCl or a similar ionic compound and you will have to test how low you can drive its concentration before the protein crashes out of solution. In general, 100 - 150 mM NaCl is a good starting point. If you can take it lower, than you should. If you need it to be higher than 200 mM to keep your protein in solution, you might have to try substituting it for other salts or for glutamic acid or arginine solutions, which also help with protein solubility.
The buffer concentration should be low as well, 10 - 20 mM is generally low enough to allow the buffers in the crystallisation condition to alter the overall pH but still high enough to keep your protein solution buffered on its own. The pH you choose should be 1.5 to 2 points away from the pI, closer to neutrality than an extreme of pH (e.g if your pI is 8, go for pH 6 and not pH 10). If your protein is happy in your first choice of buffer but doesn't crystallise, it's worthwhile trying a different buffer that works in the same pH range (e.g. both TRIS and HEPES will buffer between pH 7.2 and 8.5)
5% Glycerol also helps with solubility and, particularly, with keeping your protein stable if you intend to keep a stock of it frozen (protein frozen at -80oC can last for years, but may only last a couple of days in the fridge at 4oC) . However, glycerol tends to interfere with protein nucleation. This is useful if you're getting many small crystals in a drop and you want to get only a few larger ones instead (set up the same condition but add 1 - 5% glycerol to it), but in general it might simply stop crystals from appearing in the first place. Therefore, try to avoid glycerol as a protein additive if you can (many proteins will freeze and thaw ok without the addition of glycerol). Once your crystals have grown you can obviously try 10 - 20% glycerol as a cryoprotectant for freezing them, but that is a separate issue.
Finally, if your protein has several cysteines, you should also add 1 - 2 mM DTT (or TCEP, which is better but more expensive) to the protein solution to stop the protein aggregatating by forming random disulphide bonds between monomers.
@Ronnie and Antonio...Thanks for your kind valuable suggestion. I will keep in mind before settling crystallization.
@ Biao Quiu....Thanks for your reply.
Basically in any crystallisation process, the primary requirement is that the substance must be in solution and then the crystal should seperate out. In case of protein if you have a buffer having a pH value equivalent to its pK value, then the protein will precipitate and not crystallise as there is no charge on it. Similarly the salt solution ( suitable substance having electrovalent bonds only) will give an ionic environment which will keep the protein in solution and allow it to crystallize.
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