I am planning to set up a co-crystallization trial for my protein. I am confused as to what parameters should we consider in terms Km or Ki in order to decide an optimum concentration of substrate/inhibitor that can be used with the protein for the experiment.
Also, between substrate and inhibitor, which one should be preferred for co-crystallization ?
Hi Deekshi,
For co-crystallization purposes, you should probably be concerned with the ligand's Kd (dissociation constant) as opposed to Km or Ki. In general terms, you can use the Kd value to calculate how much ligand is necessary so that the binding equilibrium will favor some high percentage of protein+ligand complex instead of free protein and free ligand. If your ligand is cheap and/or plentiful and/or very soluble, then in practice you can add many-fold molar excess of ligand in an effort to saturate the protein. Often, however, interesting ligands are neither cheap nor abundant nor particularly soluble, in which case you may be limited to how much you can add to the protein solution. In the worse case, I hope your ligand:protein molar ratio will be >= 1. Unfortunately, there is no ratio that guarantees that you will be able to co-crystallize the ligand-protein complex; you usually just have to screen many conditions and ratios until you find one that works.
As to whether to try to co-crystallize the protein with a substrate or an inhibitor, I guess the answer chiefly depends on what biological question you're trying to answer. However, co-crystallization with an actual substrate is often very difficult, because the enzyme will be working to convert substrate to product at the same time the crystallization trials are occurring. If the enzymatic mechanism involves substantial structural rearrangements, this could impede successful crystallization. You might search the literature to see if any substrate analogs are known - these are species that are chemically similar to the actual substrate but are incapable of being turned over by the enzyme.
Just adding to Matthews answer previously, to have a theoretical occupancy of the binding sites of 95% of the proteins you need to have your ligand at a concentration 10x the Kd. This is usually the minimum concentration you need to be able to properly see your ligand in the structure, although one can always get lucky with lower concentrations..
As an example, if you have 50 µM protein concentration in the drop, and your Kd is 5 µM, you need to have 10x5+50 µM = 550 µM of you ligand in your co-crystallization setup. If you have an affinity in the low nM range, you thus basically just need to have a 1:1 molar ratio with you protein..
Hi there,
If working on simple michaelian model of one enzyme converting one single substrate into product, Km is always an overestimate of Kd therefore if work at 10Km at least 90% of the sites will be occupied.
As Matthew mentioned, in the case of an enzyme that reacts with one substrate, if you attempt to co-crystallize the enzyme with its substrate, you will most likely end up with the product rather than the substrate.
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