 Good morning, so we were discussing protein ligand and protein-protein interactions using NMR spectroscopy. So in the last class we started to understand what are the NMR parameters that can be used for understanding the protein-protein or protein-ligand interaction. We started with simple analysis of line width, the line shape, the intensity and the resonance frequency which can gives the idea about the protein-ligand interaction. Then we went ahead and we dissected the protein-protein-protein-ligand interaction into two parts where we are detecting first on ligand and then we can go and understand the detecting on proteins. So on the ligand detected experiment I started with STDNMR, saturation transfer difference where basically you can find it out binders it is a high throughput NMR techniques for fishing out the binders of a particular protein and then I discussed the line width how the ligand can have a sharper line upon binding the line width can increase so that itself can tell about which are the binders if they are binding to a protein or not. Then also like we discussed what NMR can be like STDNMR can be used for so it is a high throughput techniques. So we are going to discuss little more detail how STD can use for finding it out the binders and quantitative parameters of the binders. So here we are directly observing the ligand that is binding to membrane protein in a living cell that is what we are going to do is advanced version of STDNMR called STDD. Saturation transfer double difference right so double difference we are going to do. Discuss now STD we understand to just to make you more familiar in STDNMR what we are doing. So here is a binder here is a protein they are binding binder like a small molecule ligand has its own resonance protein will have its own resonance. We are going to saturate the signal exclusively from protein if this guy is binding the small molecule is binding the effect of that saturation will be transferred to the ligand and when we take a difference spectrum when it is not saturated or saturated far away and saturated on protein when we take a difference the epitopes that binds to the protein will be illuminated that is the STDNMR saturation transfer difference. Here we are going to now discuss the saturation transfer double difference right so what are the double difference and how you can be used for understanding the intact cell protein. So like you know like as a biologist you know the all protein cannot be purified right and purified to high concentration and all those. There are various intrinsic problem with protein purification and instability. Now still there are some proteins which cannot be taken in solution but they can be taken in a in a in a membranic environment but again the reconstitution and all those are going to be really really tough right. But those are main targets like one of the example is GPCR G protein coupled receptor they are the like a major target for drugs because they are receptors for various molecules. So about 30 percent of protein are membrane protein membrane bound protein or integral membrane protein one of them is GPCR and these are important drug targets. So if you cannot understand their binder then we are losing out lots of things so can we come up can NMR can come up with a technique where we do not need to purify protein but still you can investigate what is the binder how we can investigate that was again offered by the mayor group and that was published about 15, 17 years ago in Jax this technique called STDD and STDD saturation transfer double difference right. So now we are not going to isolate protein we are taking them in their natural membrane environment and try to understand how they are binding. So NMR has to characterize at an atomic level the binding interaction of a cell surface protein in a living cell and that is implemented as this double difference technique. So how we are going to do? Here is our whole cell right and here is a receptor and in this receptor some binders are there right. So they are going to bind here so binding reaction is K on versus K off right. So these are the binders here is a free ligand and upon it is binding. Now we are going to selectively saturate the receptor so receptor concentration of course will be very less so we have to choose a region typically methyl region where these receptors only gives the signal and we can even increase the saturation duration such by increasing the duration of this pulse or strength of the pulse and then when they bind the effect of that binding like effect of this binding can be eliminated by saturating this receptor pulse. So if receptor signal are saturated it will transfer to the binder which is small molecules and that we are going to elucidate it in the STDD NMR. So typically experiment is done like this we have a cell suspension and we have cell suspension we are taking a append of sample A will contain cells, cells and ligand and in sample B we will have only cell right. So this is the spectrum we are recording the spectrum of A where we have a protein signal like our cell signal and the ligand signal so cell and ligand is here a spectrum of D where we are doing STD of cell only and then we are like a then we are going to take a difference STDD. So here we have saturated the cell look at the effect of those cells saturation on ligand here just we saturated the cell and then we took the difference of these two. So now if you look at there are some things getting eliminated if that is getting eliminated it says that there is a binder which is which has bound and the signal the sharp signal that is coming here are from those binders the ligand that bound. So this is the saturation transfer difference to emphasize again we have taken a cell suspension split it into two in one we have added the ligand in another we have not added the ligand. In the first sample we did a STDD NMR like saturated the protein cell signal or protein signal and then without saturation we also did that we recorded the STD NMR for first one similarly we recorded the STD NMR for the second one we took the difference of those two so spectrum first minus spectrum two the illuminated signals are coming from this double difference STDD and that is telling that ok these are here as the ligands that is binding. So even without purifying protein taking from cell suspension we can find it out the ligand that can bind to particular receptor right. So that is the STDD NMR. Now I will briefly discuss to you group epitope mapping by saturation transfer difference. So group epitope like I was talking about the epitopes that binds. So basically what we are going to do is identify a segment of ligand that directly interact with the protein or receptor right. So the same concept little bit trick we have to do same concept of the STD suppose this is my ligand and this is the receptor. Now receptor is a bigger molecule ligand is a smaller molecule we are again saturating on the receptor molecules and we are looking at the effect of that saturation on the small molecules. So receptor and protein are in exchange KON and KOFF right. So here is a free ligand and here is a receptor ligand so receptor protein complexes. So we saturated essentially the receptor. Now in this experiment we are going to increase the saturation time. So the closest one will first enumerated once we increase the saturation time the now the second one the second one which is closer here the third one this is the third this is second and this is the first. So now not only we find it out which is actually interacting but also find it out how far they are from the receptor and that is called actually the group epitope mapping. So by increasing the saturation time recording the various sets of STD enumer we can find it out how where they are positioned where they are positioned in the receptor binding site. So typically these are the pulse sequence so to have a simple explanation of these for like a here you have a for D2O sample you can have pulse of saturation. So this is relaxation delay D1 typically you start with this wait for some time magnetization to come in equilibrium then we selectively using a pulse terrain we are selectively saturating the protein signal and then we are exciting it like this is a 90 degree excitation pulse then we are using a T1 row filter and finally we are detecting at T2. So during this like all the signal coming from the other interactions will be refaced and essentially we are seeing the saturation effect for D2O if you are doing in water we need to also saturate the kill the water signal so this is done by one of the water suppression technique that we have discussed earlier. So and remaining will be suppressed by the gradient so use of this selective water saturation pulse with gradient we can kill the water and rest remains as it is like STD. Saturating on protein signal detecting on the ligand signal and we have to do on resonance and off resonance in the interleaved manner right. So what is on resonance like when we are saturating the protein signal exclusively off resonance when we are not saturating protein signal so our irradiation is somewhere far right 20 degree 20 ppm or so the T1 row here we have added again to remind you it is for filtering out or removing the protein signal that might come in this. So T1 filter ensures that we are only detecting the ligand and effect of the saturation on the ligand that is what actually it ensures. So this is typical pulse sequence that we have for D2O sample and for water clean is important so we kill water using this 3919 pulse right. So here like for an example here we have only protein you can see really really broad signal so we irradiated the protein at minus 2 ppm that only protein can have. Now if you add this T1 filter you can see now the lines becomes really sharper right so you only we are getting few peaks and one day with T1 row filter you can see the lines are really coming out. Therefore if we are studying the complex system like this just like here we probably need to put this T1 row filter that defages the protein signal just filters out the protein signal and we can get a sharp line which is shown here. So protein here irradiating protein at minus 2 t 1 D with T1 row filter you can see already we are seeing sharp signal and 1 D with T1 row filter you can really get a beautiful sharp spectrum coming from the interactions right. So next is like we find it out epitope we also find it out the position of different epitope how closer or how far they are this the how high position is important because now as we discussed your medicinal chemistry friend can come and start modifying it doing the molecular like a jugglery just to increase the binders increase the size of binding so that we can tune it tune the binding property. So that is helped by STD and STDD and group epitope mapping. Now next is can we find it out because that is what we are saying can we find it out KD of the binding yes we can do how this is the simple experiment. So now this is the simple experiment that we are going we are increasing now ligand concentration and ligand concentration can be increased and then STD amplification factor how many times we can get the more STD signal that can be detected. So with concentration ligand and STD amplification factor we can find it out the KD of this so how STD amplification factor helps so we know the I0 that without anything when I sat when we saturated and we divide by the I0 multiply with the ligand excess how many times ligand was excess using these we can fit this curve and that gives us the KD dissociation constant. So STD amplification factor it is possible to quantify the active ligand concentration which allow to estimation amount of protein needed for STD experiment and it is also a specific of the ligand which has STD amplification factor of 10 or so so using some of these techniques we can probably get kind of the STD amplification factor how many times STD and this also opens an avenue on finding it out the binding strength. So here one can find it out with STD amplification factor like we discussed these guys we discussed this binding so how far or how close they are what is the STD amplification factor like this molecule was binding so we did various ligand concentration of STD ligand to protein ratio we put it the how the signal is getting saturated of different atoms H1, H7, H5, H4 and methyl we plotted the signal intensity with respect to ligand concentration and we calculated the STD amplification factor while they binding to HSA you can fit it STD amplification factor alpha times of the STD ligand concentration and one can determine KD. So essentially for each of these moiety using STD amplification factor we can find it out one of the idea we can have the idea of a KD how strongly or how weakly they are binding. So this simple experiment helps us in getting the signal. Now can we also go advance version of the STD right what I mean STD were just basically the 1D experiment where we were recording 1D can we combine this with a 2D we can get even more information. So yes STD can be combined with Cycose or even Toxy like we can get even in 2D fashion so that even our ligand becomes bigger we can get some information. So here is one example when we have combined one can combine STD with a Toxy. Now you can see here different peaks you can identify in Toxy region and since we have done STD so you have to record 2 experiment Toxy without irradiation Toxy with irradiation or Toxy on resonance irradiation Toxy of resonance irradiation and take the spectrum you plot with a various ligand concentration calculate the STD amplification factor and one can see for various epitopes the STD amplification factor can be different and you can again the map the group epitope very nicely and also possibly you can get some idea about the KD of this right great. So that is the extension of STD NMR saturation transfer difference NMR. Now just to again revise you a little bit here is my receptor molecule here is ligand molecule that binds this is non-ligand which is not binding they are like in equilibrium K of K on binding non-binding so binder binds and non-binder does not bind. So we have a KD ratio K of by K on or you can write this K on and this you can write as a K of this will be K on by K of right and we have selectively saturated the protein in the STD fashion we took the difference where there is no saturation here there is a saturation so some peaks have lower intensity so this is no saturation called off resonance and saturation called on resonance took the difference here is a difference spectrum. So now we are getting the signal from the epitopes that binds so that is a STD NMR. So now to sum up a ligand based NMR experiment screen to determine in a quantitative manner which compound binds to protein in the context of drug discovery we can map the ligand a more advanced example to use the NMR for direct characterization of protein inter like a protein ligand interactions at the molecular level through the identification of important ligand commodity one can find it out and one can determine the dissociation constant between the protein and ligand where we are taking the ligand at a various ratio we are plotting the STD amplification factor by fitting it that one can find it out the KD dissociation constant between the protein and ligand that is what we can achieve using various STD experiment. The next technique that can be used is called like a transfer NOE protein ligand complexes that is called transfer NOE. Here again we are mostly detecting on the ligand side so two techniques already I explained you looking at the line width which is determined by the relaxation property the next we talked about STD and it is a variations STDD group epitope mining and how we can use for KD determination. Now the third techniques is transfer NOE this is applied to a system where it is in fast exchange so the ligand and protein is in fast exchange KD is typically 10 to power minus 7 mole and K of rate is like a the T1 inverse here we are going to absorb the NOE because the NOE between the ligand and protein that is a transfer NOE. So we have to collect standard 2D NOG experiment like we can do 2D NOG experiment where ligand is added to the protein molecule and if there is a transfer of signal coming from the protein that is transfer ligand we can detect it. So ligand shows a signal set regions averaged over bound and free state here also ligand should be more like access relative to protein we record a series of NOG experiment and we can find it out. So a strong NOE developed in a complex is transfer to free ligand state and one can measure the free ligand resonances it is a it is even applicable to a mega like a molecular weight which is large kilo Dalton several kilo Dalton. Observe NOE can be used to determine the bound confirmation of ligand like if suppose this is ligand this is protein and we are getting the NOE from the ligand to protein we know that distance. So distance is kind of measured here and that distance fix the orientation of the ligand in the binding pocket and here the sign of NOE cross peak like will be opposite and you can get looking at that this is coming from the transfer NOE. So here we have a ligand which is small molecule protein big molecule we have a long tau strong NOE and when it is in free it will have short tau big NOE. Now when we record the spectrum of protein ligand complex one can see that these peaks which are coming here it is of negative sign and when the positives like when there is a color change positive peak sign negative peak in green. So the sign change of the cross peak indicate the binder and if they are not binding they will not bind. So here one can see here for the these guys the peak sign is changing. So here a protein ligand is happening some of these you can see these are not changing. So if the sign of the peak has changed that means it is binder they are coming from the transfer NOE experiment. So now since we find it out what are the binders you can use this for getting a protein ligand complex. So protein ligand complex can be determined by transfer NOE. Since now we have established the NOE connection between protein and ligand we can fix the orientation of these ligand in the protein complexes and using few of the experiment we can dock it and create a dock model which will tell that how these proteins are binding. So one can illuminate some of these residue that are in context residue from protein that are in context with a ligand that is how you can create a protein ligand complex using transfer NOE experiment. Right. So next experiment that I will be discussing is a water loxie experiment. Water loxie as name suggest it is a water ligand absorbed by a gradient spectroscopy. So here the concept is like there is a protein and there is a ligand but in between there is a lots of water. What kind of water? Some bulk water, some biological water. Biological water which is in vicinity of the protein molecule. So water loxie is used extensively for screening again the protein ligands. It is again a high throughput techniques and it is a reliable method for identifying small molecule. The requirement is that the binding affinity should be from micro molar to millimolar. So what we are going to do here? So assumption is there is a ligand which is binding to protein and in between there are lots of water. So and those are in exchanging. So what are exchanging? Bulk water, bound water is exchanging and then protein when it is binding that is also exchanging with the water present here. So exchange of water that is what its name is. Water ligand exchange that is happening and we are observing using gradient spectroscopy water loxie experiment. So actually it is ligand absorbed and like we are observing the ligand. It is NMR method that absorbs the ligand. It is widely used for study protein small molecule interactions. Here in this case we are irradiating now water. So this irradiated water, what is, so there is a like a nuclear overhouser effect and chemical exchange that is happening with the bound water, the protein and the ligand. So here transfer of magnetism between water molecule with protein and small molecules happens by a nuclear overhouser effect because they are in close proximity. So if they are in exchange and they are in close proximity when we irradiate on water molecule the irradiation can be transferred to protein molecule and then eventually can be transferred to ligand molecule. That is what essentially becomes active. Irradiate water gets transferred to protein, gets transferred to ligand if they are binding and that is how you essentially understand using water loxie experiment. So pulse sequence is essentially simple. So here you excite water and then mix so that the magnetization of irradiation is transferred and finally you suppress water, look at the signal that is coming from the ligand molecule and effect we are seeing on the ligand molecule. Typically we look at the small molecule. So few things you have to take care that pulse length is correct. So if you have a correct pulse length you are basically getting to get a good line shape. If you start with incorrect pulse length you see the distortion phase. So now you can see the sharp line. You can assume that sharp line is coming from the ligand molecule. Traditionally it is like a plotted negative for like when there is no water loxie signal when there is no transfer. So that does not mean that this is giving negative peak. It is plotted like that. Now we do water loxie here. Now you can see that in presence of the protein molecule you see few of these peaks. So in case of BSA you see the C and D. So these are basically with correct and incorrect pulse length again you can see it. So essentially you have to compare these two and you can see now the peaks are coming here and these peaks are coming because the transfer happened. So water molecule in water loxie a small molecule that don't bind to protein molecule does not give a positive NOE signal with water while protein will display the negative NOE because that is how it is plotted. And population that are bound to protein they will display the negative NOE signal. So that is what we are seeing. So now the experiment was done with one molar of tryptophan and yeah in presence of BSA 100 micro molar 1 millimolar of ligand signal 100 micro molar of protein signal experiment was done and NOE was observed on the ligand and this says that they are interacting and that is how you can find using water loxie. So here I will end it with water loxie. So these three four techniques that I discussed where ligand observed the line width detection the effect of T2 the STD NMR and some variation of STD NMR STDD then we looked at the transfer NOE and finally water loxie. So these are the four commonly used techniques that are there when we observe ligand. Now the next class onwards I am going to discuss the protein detected experiment which can be used for understanding the protein ligand protein protein interactions. So here I sum up and I close here I look forward to you with lots of questions if you have any doubt do not hesitate to write to us. I will be happy to answer all your questions for protein ligand interaction where we are detecting on the ligand molecule. Thank you very much see you in the next class.