I have two elements that bind to either the same or different regions on a protein. I am wondering how one would go about figuring out which of the above is the case? Is there a way of isolating the protein bound to the region and then figuring out if it is different from the second element bound to this region? Any ideas would be greatly appreciated. Thanks in advance.
Title edited for clarity.
Colibri
General Questions Moderator
You can measure the binding of each somehow?
If so, do the two elements compete for a single binding site, or not?
-Measure the binding of mixtures and see if each compound inhibits the binding of the other.
There are more complicated answers, but I think this is the one you seek.
There are a few different things you could do, but it would depend on the actual biochemistry of the protein, the substrates and the complex they form. An analytical biochemistry website or textbook could help you out.
If you know that each substrate reacts in a one-to-one with the protein, then you could take a solution with a known amount of protein in it, add a saturated amount of substrate A, and then a known amount of B, ideally equal to the amount of protein in the solution (depending on the chemistries, you might need to filter out the complex before adding B). Then filter out the complex again, and measure the amount of B left in solution. If B reacts at a different site, then the amount of B left in solution will be (within analytical accuracy) the same amount that you put in. If there is no B left in solution, then you know that it either reacts at a different site, or reacts preferentially to A at the same site. Measuring the amount of A or B in the final complex could give hints as to whether they share a site or not.
Trying to obtain a crystal and studying that is another possibility, but it’s nearly impossible to do with most proteins and substrates! Using an analogous substrate, that will permanently bind to the protein could work too - if you use A* which will bind to the A-site and not be removed, then you can do a test like above and see how much B is taken up by the protein… none would indicate same site, and some would indicate different site, etc.
So much depends on the actual protein and the substrates, though. There is no single answer to any of this.
I’m sure there are a few more things you could try along those lines, but it’s been a while since I even thought about analytical biochem, let alone studied it!
Also, if your elements have spin > 0, then presumably a HOESY, HETCORE or HSQC experiment. (That would be NMR). You’ll have to have the correct pulse sequence programed into the machine, but I’m pretty sure somebody will have it.
Keep in mind, unless the detailed full structure of the protien is known, it will not tell you where the “element” is in the protien, it will only tell you if it is in the same place as the other element. Even that will take some major interperetation based on coupling constants and whatnot. Still, I suspect anyone with real experience in this area would be able to figure it out. The coupling constants of all the elements are known and in some cases the experiment itself eliminates them.
I think the trouble with this scenario, is that it assumes that reaction with A only inhibits reaction with B if they react at the same site. The two reactants may very well react at different protien sites yet still effect the reactivity at the other site. The only sure way to explore the site reactivity of each element is through direct spectroscopy of the protien both occupied an unoccupied.
I do not know exactly what is available to the questioner. If you do not have an NMR facility available to you, then my plan is useless. I specifically mentioned several NMR experiments which could be used to deterime if these “elements” are coordinated to the same spot in a protien. All of my methods assume that the “elements” in question have a spin > 0; however, assuming that is the case, there are basically an infinite number of NMR experiments that could determine precisely what the OP requests. Any experimet utilizing the heteronuclear nuclear overhouser effect would be sufficient to determine what the OP wants. I think, that any reasonably funded laboratory would do exactly what I suggest, but I am not a biochemist. I have just enough experience with NMR to know that any experiment employing a heteronuclear overhauser effect will do exactly what the OP want assuming you have somebody to interperet the spectrum.