What is the critical concentration of a ligand that is used to cocrystallize a protein along with its ligand and how the varying concentration of the ligand effects the crystallization.
The idea is to saturate the protein's ligand binding site with the ligand. The ligand concentration must therefore be at least as high as the protein concentration. If the Kd of the ligand is higher than the concentration of protein in the crystallization, then the ligand concentration should be higher than the protein concentration. If possible, try to use at least 10-fold above the Kd case to achieve near-saturation.
Thank you Adam....
If ligand have the least solubility i.e less than the Kd,then how to use higher concentration ?
Low ligand solubility is often a limiting factor for co-crystallization. The crystallization conditions (especially pH and ionic strength) can affect ligand solubility, so if you can try different conditions you might be able to improve ligand solubility. Low ionic strength improves solubility of hydrophobic compounds. Ionization of the ligand improves its solubility.
Also, ligand binding to the protein lowers the concentration of free ligand, so increasing the solubility of the ligand overall.
You may consider adding your ligand once the crystals have grown. 2 potential advantages :
- You will spare ligand.
- The ligand may be more soluble in the crystallization condition.
I like the 1:10 for protein:ligand ratio. Importantly: before mounting crystals, you would like to back-sock the crystals in a solution with no ligands. In any case, things would greatly depend on the affinity of your protein for the ligands.
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