 So, welcome to today's lecture where is discussing important topic of protein ligand interaction and how you can use NMR spectroscopy to understand this. So, in the previous class we looked at a very simple technique that is based on polarization transfer can be used for quick determination of a ligand which is binding or which is not binding to protein and that can be used for a screening of ligands and essentially this is high throughput technique for pharma industry to find it out what is the binding epitope whether this ligand bind to protein or not where actually on ligand site it binds to protein. That is a very simple technique but as I said let us take it to the next level and we find it out how we can use this technique for finding it out not only soluble protein also integral membrane protein or a protein on the self surface because you know the membrane proteins are one third of the total proteins and they have very, very important functions like they are involving signal transduction we discussed briefly this last class like GPCR. So, G protein couple receptors and the GPCRs are one of the primary target for drugs. So, many drugs that are discovered or designed just for the GPCR targeting. So, GPCRs are very important then pumps like molecular pumps like calcium pump potassium pump and all those or ion channels these are all membrane protein and they are also very, very important for many pharma industry. But they are difficult for solubilizing and therefore in a typical conventional way the protein ligand interaction becomes difficult because they are membrane invaded. So, their natural environment is around lipid so they cannot be solubilized and they cannot be crystallized therefore crystallography is also difficult to find protein ligand interaction in this case and this is primarily because lipid is their natural environment. Now one can crystallize one can solubilize but you need to have a lipid for these molecules. So, that becomes a project in itself here for pharma industry we need to find a target not getting into a structures. So, if you look at actually genome encodes one like almost I would say 30 percent of proteins but structure knowns are very less. So, membrane proteins although it is important target for pharma industry but structurally very, very limited knowledge we have compared to other solubil protein. Therefore, suppose you want to quickly get into this drug determination and you do not want to get into this structure solving process which takes years to solve a structure. You can use these techniques that we were discussing quickly to identify and binding ligand. So, how we do that that we will go to this technique which is called SDD we will discuss today. Here we are going to do direct observation of ligand binding to membrane protein in living cell by something called SDD NMR saturation transfer double difference. So, how we do that that is what we are going to today discuss. So, now we understand why it is difficult because membrane proteins are not soluble their lipid is their natural environment. So, we cannot do conventional SDD NMR but these are important targets. So, therefore we need to understand this and we can implement this binding by saturation transfer double difference. So, here is just a schematic suppose this is my cell surface and here is one receptor and this is environment for this protein receptor some part inside the cell and some in the membrane and some is extracellular domain. Now, this extracellular domain suppose we have a binding pocket where my molecule is binding. So, there is a binding site here and in this site the molecule like Cypher, Rg, D, C, B, F some molecule which binds. Now, this protein has protons this ligand has protons they are binding and going off. So, micro molar binding is there K on and K off is happening. So, now here what we are going to do now is same concept we are going to use it. So, this is the sequence saturation of protons on the protein side here receptor side. So, we are selectively saturating the signal on the protein or receptor side and looking at the effect of that saturation on the ligand. So, suppose you increase the saturation effect what will happen? The ligand which is the ligand proton which is quite close will be more perturbed and which is little far will be less perturbed and so and so far. So, now if you keep increasing this saturation the effect will be transferred in a distance. So, this will be like whatever is nearer to binding site will be more perturbed then this and then this if you increase this this will be perturbed this will be perturbed but this will be less perturbed. If you further increase all of them will be perturbed and that is what you can find it out the protons or the epitopes which is closer to the binding site which is little far to the binding site. So, experiment is simple in saturation transfer double difference you do not have to do much you just take a cell suspension. Just I forgot to tell you this method was developed by again the Meyer group in Germany and that was published in Jax some 14 years back. So, what we are going to do here in this experiment we are taking a cell suspension we are splitting it into two. So, this is a pent up where we have cell suspension and this cell suspension has our like membrane embedded protein or protein expression on the cell surface. So, here you can see this is solution these are all cells cell suspension. So, all this integral cells are there we are splitting into two sample that has only cell sample that has ligand and as well as cell. Now, here same thing we are doing spectrum of STD spectrum of cell and ligand we are doing STD that means, so let me explain again here STD we are going to do on both. We have total cell suspension we divided into two one where only cells are there another where cell plus ligand are there. We are going to do two STD experiment one with only cell suspension another with cell with ligand. So, here in this STD experiment we put up somewhere on minus one or so and look at the STD difference. Then here again we put up at minus one ppm or so and that effect will be visualized on the ligand. Since there are this is a point heterogeneous mixture so we do not know because ligand is not very clean. So, therefore, ligand has signal which is here also as well and cell suspension has all the signal coming from the proteins. So, if you take a difference of this STD where cell plus ligand was there and STD where only cell was there take the difference that is called saturation transfer double difference spectrum A minus spectrum B. You get few of the peaks that are actually shown in this spectrum. So, if there is no binder ligand you should get essentially zero signal because there is no saturation transfer. But if you are getting these peaks that means there is a binder and these binders peaks has like this. So, now you can record only ligand signal and you can find it out which protons in the ligand are actually interacting with this receptor. Just by this simple experiment one can find in whole cell that whether there is a binder or not. So, just again to summarize this is a little complex technique. What we are doing our target is a ligand for the receptor which is on the cell surface. For this we are doing experiment called saturation transfer double difference. We are taking the whole cell suspension. We are dividing that into two, one only with cell, one cell plus ligand. We are going to do two STD, one for sample number A, one for sample number B. Now, this two STD are subtracted if you are getting the signal in the difference double difference spectrum we identify that this suspension has ligand which actually is binding to the receptor. Now we can record the ligand spectrum and we can find it out the atoms that are involved in the interaction and that is how you can find it out in STDD that what is the binding mode. Now, let us go further and this is called this experiment is called group epitope mapping by saturation transfer difference NMR. What we want to determine this segment of a ligand in direct contact with a protein or receptor. So, suppose protein is big anyhow and ligand is also big so all the moiety in the ligand is not interacting with a protein some moiety is interacting and that is what we have to find it out that which moiety is in proximity of this. So, essentially briefly I discussed it but let us go in detail. So, here is our receptor ligand complex receptor is big ligand is small and ligand has many protons. We are selectively saturating on protein and looking at the effect of that saturation on the ligand. The only condition is that they are in micro molar binding range so K i and K off is there. So this is free ligand. Now we are doing several experiment we are increasing this saturation as strength. So, if at low saturation only direct contact protons will be shown in STD. If you increase saturation this will be shown then further increase this will be shown. So on increasing saturation you can find it out how the protons are getting affected by this transfer and this can identify the binding epitope even for the complex ligand very quickly. So, let me repeat it again here is my receptor this is my ligand. So suppose this these two are my moiety of the ligand so this binds and this does not bind. If I increase saturation first protons from here will show STD effect but if you keep increasing slowly from protons from here will also show effect STD effect. So we know that this is closure and this is little far and that is what is group epitope mapping by STD NMR. So essentially this is the pulse sequence you do basically in D2O so that you remove water background you can do even in H2O but then you need to add something called this water suppression peak. So this is water gate and then you need to have a T1 row filter just to remove the protein signal. So experiment is very very simple here is a saturation then you have a 90 degree pulse and then T1 row filter for removing the protein signal then you detect it. Now you have to increase this saturation time so if you increase the number of N saturation time will increase and that will be shown essentially on the protons of the ligand the closure one will be affected first then follow and so you have to do two experiment on resonance and off resonance where you saturate here you would not saturate and that you can do in interleaved manner and then you can subtract it to save the time. So this interleaved will save the time so you saturate it you get a STD signal if you keep increasing saturation more and more peak will be affected and that will tell you the group binding epitope. So one example here if you take a protein because proteins is huge so if you take just 1D of protein we are getting the broad signal. Now suppose you take a protein and irradiate at minus 2 ppm so minus 2 ppm is essentially some protein signal will come something from too much shielded that will be off field shift. So essentially if you take a you see this all the protein signal will be gone that means minus 2 is the right ppm for saturation. Now you do 1D with T1 row filter so as I discussed T1 row filter you can put I do not want to go in detail but T1 row filter essentially can remove all the protein signal. So here mostly we are getting now the signal coming from the ligand you can see the sharp line as we discussed previously sharp line means line from signal broad line means from complex and protein. So sharp line coming from the ligand. Now you do reference spectrum 1D without T1 filter you get a broad line and T1 1D with a T1 filter you get a sharp line. So if you do STD you get these peaks and these peaks will tell that this particular ligand is interacting. Now you can increase the saturation time and you can see the effect on these different protons and that will tell you which are nearer or further. So if you do that saturation time you can plot it the effect of that as a either ligand concentration or saturation time concentration. So by doing that one can find essentially an STD amplification factor. So what is STD amplification factor? I0 means non-saturated and here is saturated divided by non-saturated into whatever ligand excess that we are adding. So now STD amplification factor can be fitted into this and that can give you binding strength of this particular protein to the ligand. So using STD amplification factor which is also possible to quantify the active ligand concentration which is therefore allowed to estimation of amount of protein needed in the STD experiment right. So here just we are increasing ligand, ligand is in excess. So here say protein is 2 micromolar, ligand is 200 micromolar. So do we need that much. So just to find it out we keep increasing the ligand concentration and you find it out what is the optimum ligand you are going to use for this particular protein target. And now you see this is giving an important aspect, suppose we are targeting one protein. Now how much like pharma industry can also take this in account that how much protein you need to saturate this receptor or to bind completely with a receptor. So that helps in determining the dose of the pharma industry. So this ligand concentration you can just get it by simply doing STD NMR. So after some time it becomes saturated and you do not need any more ligand for that. So in a specific case the ligand has a STD amplification pattern of 10 then protein concentration is 50 micromolar and ligand concentration should be 500 micromolar. So that is what it gives you the idea of saturation. As we discussed so STD amplification pattern you can find it out essentially which protons is nearer which protons is far and you can find it out here like H1, H7, H5, H4 they have a different saturation range in a ligand to concentration manner and that can be used to find it out the essentially the KD, this is ligand concentration and this is the amplification pattern for STD. So with STD one can find it out the binding epitope, we can find it out group epitope that we just looked at here, group epitope mapping, the position of the binding atoms and we can find it out what is the saturation by STD amplification pattern, what is the minimum concentration required for saturation of the receptor then we can find it out the KD. This STD is a simple module that can be added even to a 2D. So suppose we can add actually we can add STD in Toxy so you can record one STD Toxy without saturation, one STD Toxy with saturation and you can find it out here just for an example you can see these peaks are going vanishing here so you know that these peaks or even the peaks from here one can find it out that like this peak if you look at in the reference and this STD are disappeared so not only one day you can extend this to 2D but time required will be more in the 2D manner you can find it out even the side chain like many other atoms in more resolved way which is actually involved in the binding. And similar you can do in STD Toxy manner you increase the concentration of the ligand record several STD Toxy and you can find it out KD of this binding protein ligand interaction KD you can find it out also group if it up mapping you can find it out just by fitting this curve. So essentially STD is a powerful tool so what we learn the protein ligand interaction if this ligand is interacting this is non-ligand there is a K on and K off this ligand binds to protein and there is a KD K off by K on. So now what we are doing off resonance all the peaks will appear from all the ligands. Now if you saturate it the effect of this saturation is transferred to the binders and you see some of the peaks have a different intensity and you take this spectrum you can find out different spectrum and that is given by these protons of the binder that is what we looked at. So to summarize this is a ligand based NMR screening experiment to determine in a qualitative manner which compounds bind to a protein in context of drug discovery which quite rapid and you can use that as a throughput technique for finding it out binders to a protein. Now ligand mapping you can do a more advanced example we just showed use of NMR for direct characterization of protein ligand interaction at the molecular level and identification of important ligand moiety this moiety is binding or that moiety. So in a group if you took mapping one can find it out the portion of ligand we can also determine this dissociation constant KD between the protein and the ligand. So now little bit more than STD there is another method which can be used for protein ligand interaction this is called transfer NOE transfer NOE you know nuclear overhouser effect. Now transfer NOE means we are transferring the NOE from ligand to protein to find it out whether this ligand is interacting with protein or not. So this transfer NOE method is also used and this is again saturation transfer based method so for ligand protein interaction. So only thing it has to be in fast exchange and KD should be in quite a strong like 0.5 or greater than 1 micro molar and then K of also should be smaller. One can collect the 2D nosy experiment so essentially what we have to show ligand shows a single resonance like if the protein and ligand is interacting and they are exchanging very fast shows only single set of peaks and average over bound and free form and ligand generally is in access to protein that is what we transfer NOE. Quite a strong binding that should be in micro molar range and one record nosy and then ligand should show only one set of peak because they are in quite a bit of exchange and ligand should be excess of protein. So if you do that the strong NOE will develop so now this is binding right so NOE is a distance dependent phenomena. If it is binding and all the time it is spending here so then NOE will develop from complex and that is transferred to the free ligand state and that one can measure from the free ligand resonance and that can also be applicable to large molecular weight right. So NOE can be used to find it out bound conformation of ligands. So what we are saying now my ligand is binding to protein and it is exchanging so sometime it spends in bind form and sometime in free form. Now if they are complex formation the NOE will develop between in the complex and that will be shown in the spectrum, nosy spectrum. So all the NOE that coming from complex you can absorb it and find it out the protons that are in close complex of the protein molecule. So one can find it out now by what is the binding epitope in transfer NOE manner and the so now when protein will interact with the ligand now ligand NOE sign will change because now it becomes bigger molecule. So therefore the NOE cross peak relative to like sign will change and that is what one can find it out. So essentially this is the case protein has a long tau short NOE, ligand has a short tau big NOE and if it forms the complex like here the NOE pattern of the complex will change and that is what you are going to see here. So here are all those negative NOE okay so negative NOE are green this is coming from the complex and these you can find it out here if you look at this was positive NOE upon complex formation the sign had changed so this means these are the protons that essentially are binding some of them they are not changing so that means those are not binding okay. So that is what you see and that one can find it out whether it is binding or not binding. So one can find the complex ligand and one can use this information in a residue specific manner and you can find where my ligand is binding to the protein by doing by getting information from the transfer NOE and you can use this information to create a dock model something like HEDOC HEDOC is a software which can be used for getting this complex model so you can use those information to create a complex model of binding from protein to ligand. So to sum up what we have here so STD I showed you the STD can give you the binding strength like KD also the group binding epitope you can do that by quickly but STD is not only methods again polarization transfer based method like ligand protein interaction can be also used by transfer NOE in transfer NOE what we are going to do is looking at the NOG spectrum of ligand if it is in exchange with a protein in the complex form. So because of this interaction it gets a negative NOE sign and those negative NOE can be identified and those are the protons of the ligand that are interacting with the protein you can use those information to find it out which protons are interacting with a protein one can now use this information and create a dock model using HEDOC and have a complex protein ligand complex structure. So these are very very easy simple techniques that can be used in a structural biology as well as in pharma industry to find it out protein ligand interaction. Thank you very much I hope these two lectures on protein ligand interaction will be useful for you in future for drug designing, drug discovery so and so forth. Thank you looking forward to see you in the last class.