I want to crystalize my protein with its ligand (Mw 423.5 gr/mol which is in the range of crystal staining dyes). Is it obligatory step separation the charged proteins (Pr+ligand) from uncharged proteins (by e.g. DEAE anion exchange) before protein concentration and crystallization?
I think we can use two methods: co-crystallization (Ligand addition to crystallization buffer as an additive) and ligand soaking (exposing the apo-protein crystals to ligands). But as I read the literature in this kind of proteins they used almost anion exchange chromatography to separate excesses ligands and apo-proteins from complex of them. according to our knowledge about crystal nucleation and growth, What will really happened during crystallization and crystal nature if we ignoring this pre separation step and co-crystallize them?
Hi there,
Have a go with cocrystallization first as long as ligand is in large excess in order to saturate protein binding site. You don't care about the excess of ligand present in the crystallization liquor. Soaking can be consider if no hit is obtained from cocrystallization screen and if apoprotein does crystallize...
In order to separate the protein-ligand complex from the protein without ligand and the free ligand by ion exchange chromatography, it would be necessary for the ligand to bind very tightly (nM Kd). Otherwise, the ligand would dissociate from the protein during chromatography. And for such a tight ligand, you could probably just add an equimolar amount of ligand to the protein and skip the separation step, since there would be no free ligand or ligand-free protein.
For less tightly bound ligands, crystal soaking or co-crystallization are suitable approaches.
Hello. The first question I would ask is how do you know you have a complex? What method or technique have you used to confirm complex formation?
I agree, co-crystalization is the method of choice using an excess of ligand. If no crystals appear with ligand then try crystallising the apo protein followed by soaking of the crystals. Ion-excahnge is not a prerequisite for crystallisation. Many proteins have been crystallised without this step. Consider the purity of the protein prep. If it is good then probably no need for ion-exchange. If you get no crystals then try with ion-exchange. There is never an easy way to predict what will work best so go for the simplest option first.
I agree with all my predecessors. Ideally, you want to have as homogenous (and as pure) protein state as possible and one way to achieve that is to use large excess of ligands (we sometimes use ratios as high as 50-100 molar excess). What protein to ligand ratios you can use will of course depends on ligand solubility. As suggested here, I would try co-crystallization first and if no complex is obtained I would try soaking. Good luck!
""according to our knowledge about crystal nucleation and growth, What will really happened during crystallization and crystal nature if we ignoring this pre separation step and co-crystallize them?""
Nobody can tell you what "really" happens during crystallization.
You absolutely do want to run ion exchange chromatography and SEC on a protein sample prior to crystallization screening, regardless if you worry about a ligand or not. Those two purification steps add much to the homogeneity of your sample and even easy to crystallize proteins give so much better crystals when the contents of your crystal units are more identical.
Essentially, if you purify for crystallization, never omit steps in your protocol, add steps.
examples:
1) If you want the apo protein but a native ligand binds with ~pM affinity, try refolding first
2) If you want a native ligand in the protein that binds moderately strong or a protein complex, use mild conditions first to preserve it
3) If you want a specific ligand or small molecule that binds weakly, remove any contamination that could block the binding site by combining many diverse chromatography steps and add excess ligand to the pure sample.
Also, purify proteins as fast as you can, they typically don't get better by sitting in solution.
And yes, if you have enough resources to screen the same sample several times, just try a purification that makes sense to you and see if you get crystals, that's what most people would do first.
In most screening formats you can screen two drops or more per condition so you can do apo and pre-complex in one sitting.
thank you all for your guidance.
Obviously, the apo-protein is purified perviously (in this case by Ni-NTA agarose column). Also it seems the protein/ligand affinity is moderately strong. So the co-crystallization and soaking will be the first option.
We could use SDS-PAGE of selected crystal and also differences in fluorescence properties (in this case) for understanding the protein+ligand complex in crystal. Also electron density map will given the final judgment.
- Badges
- Science method
- Similar topics
- Methodology
- Crystallography