 So, good morning, this week we are trying to understand protein ligand and protein-protein interaction. So, in the last class I explained that why protein-protein interaction is important because all the cellular communication happens through protein-protein interaction or some interaction of proteins with other molecules and we looked at different methods that can be used for protein-protein interactions. So, two important aspects where they are to understand protein-protein interaction like how, like what is the thermodynamics involved there and what kind of structural change happens. So, for thermodynamics we looked at some of these techniques that can be used to derive the thermodynamic parameter like isothermal titration calorimetry, ITC or SPR surface plasmon resonance, these are the two widely used techniques for deriving the energetics or thermodynamics involved in protein-protein interactions. We also looked at some of the structural techniques that can give the structural aspects of the complexes like X-ray crystallography, cryoelectron microscopy, these two are high resolution techniques. Other than that like a SCC, size extrusion chromatography, analytical or tracentipugation, the dynamic light scattering, static light scattering, some of these techniques can give you shape and size of the complexes that are formed. So, they also you can decipher some of the characteristics of the complexes that forms upon interactions. And I try to end why NMR, kind of like why NMR is the better techniques because it gives both these parameters, the thermodynamic parameter of these interactions as well as structural parameters of these interactions. So, NMR is uniquely placed to understand protein-protein interactions and also looked at what are the requirements for that. So, we will be starting from there, what are the requirements for the NMR spectroscopy and just to rewind a little bit what actually NMR can offer either from the labeled protein like 13CN15 labeled protein or an unlabeled protein. So, you can illustrate this structure using some restraints, restraints can be NOE-based restraints, nuclear overhouser effect that measures the through space distances between these two nuclei. You can measure the dipolar restraints using like using RDC and all those so you can like from these you can measure the distance restraints, torsion angle restraints and orientation of the bound vector. So, using all of these restraints one can get the high resolution structure of the system in solution if you are doing solution state NMR. Then we can also measure the diffusion using some of the like techniques that NMR has called DOJI where you can measure the hydrodynamic radii, some additional techniques can be used to measure the RG like MD and all those can be used and NMR also can offer the dissociation constants. So, this we are going to now discuss little more detail. So, structural details using NMR can be find at atomic resolution and then we can also get the quantitative kinetic parameters for such measurement. Therefore, we can say that NMR is uniquely placed to offer the structure as well as the thermodynamics of the protein-protein interactions. So, that is what the summary from the last slide. So, it can gives you stoichiometry with what ratio these two partner interact, what is the kinetics, what is the rate of the interaction and what are the thermodynamic parameter involved in the interaction. So, these are the some of the parameters that can be derived from PPIs. So, let us start how we can get all these parameter, what we can aim for from the signal that comes in NMR. So, let us take two molecule like a say here is one molecule and it interacting with small molecule like this now and changing its conformation something like this. So, there is a on rate of association, there is a off rate of dissociation. So, that is what happening these two conformation is changing upon interaction now so that means there some exchange is happening there and we can define this exchange phenomena called chemical exchange where two or more state, two or more state during the time of recording of NMR spectrum that changes. So, this we can define as a chemical exchange and we are going to use this term and again and again. So, it is a simple two or more state are in exchange when we are recording the NMR spectrum that is a chemical exchange. So, typically when we record an NMR spectrum you get peak like this now these peaks has few property which can be deciphered to understand what is going on upon interactions. So, if there is a free protein you get peak like this but when it forms complex you can also get peak like this and just comparing these peaks you can learn a lot about what is going on. So, let us analyze this peak in a simplistic term. So, when you record a peak what we get a resonance frequency which you can measure in hertz or radian per second or in simple term chemical shift that is in ppm. So, that is a that gives the position of the peak right the resonance frequency is the position of a peak. Next parameter what we get intensity of the peak how high it is right so intensity and the third parameter that we are going to get is a line width how narrow or broad it is. So, three parameter just by recording a simple 1D NMR spectrum we can get the chemical shift right. So, chemical shift is given by omega that is can be denoted in ppm radian per hertz or radian per second or hertz and intensity gives the height of the peaks and lambda. So, what these three parameters basically denotes in such case. So, let us see first and most important chemical shift what it gives the frequency at which it is resonating. So position of the spectrum now resonance frequency omega or the chemical shift tells about the chemical characteristic of that particular nuclei. So, when it is in free form it has one chemical characteristic when it is in bound form it can change its characteristic. So, resonance frequency may change upon complex formation. So, this is the first readout that we can have. Next one is intensity of the peaks. Now, intensity tells about how many molecules contributing for that particular intensity. So, peak height or the intensity tells about the relative concentration thus can tells about the population and size of the nucleus resonating at that particular frequency right. So, how many molecule contributes to that particular frequency that is given by the peak height right. The third parameter is a lambda the line width right. So, line width in the NMR spectrum depends upon relaxation property of that nuclei which is basically T2 or the correlation time what it tells. So, suppose molecule is like a small. So, that means it is its relaxation time will be of one kind molecule forms complex it is now slowly tumbling that is a correlation time how fast or how slow it can tumble. So, it is a slowly tumbling its peak will be broad. Now, the line width that we are getting here is telling the characteristic whether it is a small molecule or what is a big molecule. So, these three parameters just from recording 1D spectrum we are deciphering what is the resonance frequency, how many molecule contributes to it and whether it is relaxing fast or slow what is the line width. So, using these actually we can already a start understanding the protein-protein interactions ok. So, let us divide this protein-protein interaction into two parts, first part where we are looking at the protein, another part where we are looking at the molecule small molecule right a ligand. So, here is our protein P and here is our ligand L right. So, these two are forming ligand protein complex. And generally let us define it is a small molecule which can be a peptide or even a small molecule peptide means less than 10 amino acid. Now, in this case when we are understanding protein-ligand interaction we can either probe on the ligand side this one or we can probe on protein side or we can probe simultaneously both that is the ability NMR has that we can probe both of these and it can bring the information about which nuclei in the ligand are involved in the binding. So, as we mentioned like we will be dividing this protein-ligand interaction into two, one first is ligand based detection and another is protein based detection. So, for few like a few slides initially few one or two lecture we will be focusing on ligand based observation and then we will shift to protein based observation. So, let us start with a ligand based observation. So, here ligand is typically a small molecule protein is big molecule. Now, because of this ligand has some property its small molecule protein relatively is bigger molecule it will have some property and when it forms complex it will have its own third property. So, if the ligand is small it will slowly relax that means lines are going to be sharper it will has fast diffusion translation on diffusion is small molecule. So, it will be fast diffusion and it will have a positive annoying and there will be minimal spin diffusion like there are not too many spin. So, spin diffusion will be minimum on the ligand side. Now, protein since it is a bigger molecule it will be fast it will have fast relaxation like here the lines will be sharper here lines will be relatively broader it is a bigger molecule. So, slow diffusion it will have negative annoying and since there are too many spins in protein so there will be fast spin diffusion when it forms a complex now complex is a bigger molecule and even if we are looking at the ligand molecule it will have fast relaxation because now it has formed a complex right it is a tighter complex. So, the lines will be broader it will slowly diffuse it will have negative annoying and of course it is in the vicinity of many spins therefore it will have fast spin diffusion. So, these are the parameters are going to change even if we are looking at the small ligand molecule because now upon complex formation the overall size has increased. So, first relaxation slow spin diffusion negative annoying and fast spin diffusion will happen right. So, let us let us delve deeper into how we can design or why we can understand the ligand detection detected NMR experiment. So, here assumption is our ligand is unlabeled and protein is also unlabeled right. So, we are not going to isotrically label any of these this is smaller one protein is bigger one. Few of the experiment that has been developed and I am going to discuss in detail one is called saturation transfer difference another is called water log C these two experiment I am going to explain in detail. Now, saturation transfer difference as NAMS name suggested it is a difference in the saturation that the molecule can absorb right. So, that is a like a 20 years ago this technique was developed and this was widely used for throughput screening of the binders for a receptor or a protein molecule this is quite widely used. So, we are going to discuss in detail how saturation transfer difference works and how we can use for screening the binders. The declaration here for SCD it is a size ratio dependent. So, there has to be size ratio difference like our size ratio consideration for ligand and protein and another important phenomena that we have to take care that there should be no signal overlap with the protein. So, ligand should have one range of signal protein should have another range of signal at least where we are saturating it there should not be overlap then only we can understand the SCD and no spin diffusion within the ligand. So, typically small ligand should be there. So, minimally the spin diffusion should happen for SCD to happen. Now, water loxie is another technique which basically exploits the surface pocket of for binding right. So, if a molecule a small molecule binding to a receptor there is a surface of binding right. So, it exploits basically what is at the surface and the small molecule actually should tightly bind to receptor and there should be fast exchange rate and ligand should be accessed then only water loxie can be used for understanding it. So, water loxie little bit later I am going to come back but these are the prerequisites for water loxie. So, let us start with saturation transfer difference spectroscopy STD NMR that is what in short it is called. So, here is our ligand molecule, here is our protein molecule. Ligand has some protons which is resonating at delta A protein has some protons which is resonating at delta B and these two are basically in fast exchange that means their binding should be in order of loosely called it is order of micro molar range right. So, Kd should be 10 to the power minus 6. So, this is one of the prerequisites for STD to happen. There should be difference in the molecular weight. So, typically this should be Dalton, few Dalton and those should be kilo Dalton. There has to be in fast exchange when they forms complex that is a protein ligand complex. Now, saturation will be transferred what we are doing we are saturating the signal exclusively saturating the signal that is present in the protein molecule and since the saturation gets transferred it will be transferred to ligand molecule and that is what we are going to take a difference. Once we are saturating protein molecule what is the signal of the ligand molecule and when we are not saturating protein molecule what is the signal we take the difference and that will tell whether ligand is binding and or not binding and if binding where it is binding. So, essentially you can find it out epitope that binds to the receptor molecule. So, how saturation we transfer we are saturating on the protein molecule. Now that saturation can only transfer if they are they are binding so they are in close contact close proximity. So, binder will see the effect of this saturation that is happening on the protein molecule. We saturate by irradiating these resonances and that can be transferred to the protein molecule. So, as I mentioned the KD for binding should be in micro molar range and size difference between the ligand and target should be about 15 fold. So, if we are taking 100 Dalton of ligand it should be 1500 Dalton of the protein molecule or bigger even is better. So, that is how that is the one of the prerequisites another is they should have a micro molar binding. So, typically while doing experiment we take small concentration of protein which is in micro molar and ligand should be in millimolar range because we are detecting on the ligand. So, ligand generally should be high access so that we can get exclusively signal coming from the ligand molecule. Now peak from the ligand bound to protein R is not expected that even may not come and a particular protein signal is irradiated. So, what we are doing as we saw while doing the experiment we are irradiating the signal or the resonances atoms present in the protein molecule and the effect we are observing on the ligand molecule. So, so here like suppose these two peaks this is from ligand molecule this is from protein molecule you can see this is intensity is high and little bit sharper. This is the intensity is low because we have taken low concentration lines are broader. Now we irradiated the protein molecule and since they are binding the effect of that irradiation is being seen on the ligand molecule so you see intensity of the ligand has decreased and this of course has vanished. So, this was published by Mayer group in Anguante, Simi from 20 to 23 years ago and this has become really popular in industry for drug discovery and design. So, what we are doing we are selecting a signal in the protein that can be irradiated like this is a typical protein 1D spectrum you see there will be some uphill shifted peaks which can be selected for the irradiation so typically methyl peak can be chosen. Now in this case ligand will not come ligand i is going to be only in this region so we are saturating one exclusively protein peaks and since they are binding we are going to see the effect here. So, this is the typical pulse sequence we irradiate selectively so there has to be a selective pulse a pulse terrain that we can have that selectively we are irradiating on protein signal and here the ligand signal are absent and then we are detecting like mostly on the ligand signals. So, this is the reference 1D spectrum from the ligand and now we did STD we irradiated protein this is the STD spectrum so then we take a difference of these now we can get the basically some of the peaks that got lower intensity so that means STD is happening and if there is no STD happening there will be no signal. So now where there is a effect is seen intensity will reduce for the binder for nonbinders when you take the difference between the reference and STD and MR the signal will vanish so that means those are nonbinder right so that is what you get in STD. So let us take one of the example so suppose this molecule is binding to a protein binding to a receptor we take the proton NMR you can like you can find it out all the peaks that are there A B C D E you can find it out then we did STD NMR saturated on the protein and detected the effect of that and then we took the difference of non-saturated versus saturated how do you do not non-saturated you saturated minus 20 ppm or plus 20 ppm where there is no resonance and when you are saturating you are saturating at the methyl peaks and you take the difference of these two 1D spectrum. One case we saturated somewhere here minus 20 ppm another case we are saturating here these two 1D subtracted and upon subtraction whatever peak is being illuminated is seen here. Now you can see we are seeing only say out of those many resonance we are seeing only D E B and F right D B so that means here D E F and B so these are the atoms that are binding to the receptor or the protein. Now not only we find bind like a binders this is the binder but in the binder which are the atoms that are binding. Now this is a great help right when we are doing high throughput drug discovery we are starting with a 500 compound just by recording two 1D NMR in few seconds we find it out binders what are the binders so that gives us lots of clue. Now our medicinal chemistry friends can come and they start modifying few of these groups and make it a stronger binder or weaker binder depending upon what is the necessity. So medicinal chemistry can be invoked here to make it better binder or weaker binder. Now epitopes is very important for drug optimization the binder optimization and that is what STD NMR offers to us. So like even like there are if suppose there are like a stereo specific questions comes or isotope sorry this isotope isomers comes in that case also you can find it out which is the binder. So for an example let us take two of these compound six methyl tryptophan and seven methyl tryptophan they are suppose binding to a protein molecule right. So we have a mixture of this and we recorded a 1D spectrum we can assign these so red peaks are coming from seven methyl tryptophan and blacks are coming from six methyl tryptophan. We put the protein receptor for these we did STD NMR like we saturated on protein at where there is a signal of protein and where there was no signal of protein we subtracted these two spectrum and here we got an STD spectrum. Now you can find it out like just by comparing that the black one peaks are available that means six methyl tryptophan out of these two was a binder and seven methyl tryptophan does not matter. So just a simple experiment where the human serum albumin as a protein was chosen and this six methyl tryptophan and seven methyl tryptophan out of these two were able to differentiate the binders right. So corresponding STD NMR you can find it out which is the binder which is the which is not binder great. So that is a that is a great tool for drug discovery. So just as a schematic here I am showing we have taken a protein target and we have mixture of ligand just for cartonistic representation I have taken here the square shape sorry rectangle shape the oval shape and the diamond shape ligands and we are doing now drug discovery which is binding right which out of these which ligands is binding. So today we recorded the STD NMR like here is a reference NMR here is a binders NMR and we figure it out that these oval shapes has binding to the receptor or protein target not the others. So you can even change the saturation time and you can find it out by doing couple of more 1D spectrum how better they bind. So that we will be discussing little more in detail right. So now that was the critical NMR parameter that can be like a critical NMR experiment that can be used for analysis of the binder. So now NMR analysis of protein ligand interaction like a ligand line with as I said when they are in free form they will have sharper line and when they binds since they are some tumbling slow the line will be broader. So as we have seen before line width directly related to molecular weight so upon binding the line width will be broader. Now so yeah a small molecule is order of magnitude lighter than a typical KDA and therefore they have sharper line these guys will have broader line and small molecule has generally sharp line protein has a long broad line. So small molecule binding upon a protein now lines become really broader and just looking at the line width which is given by T2 one can find it out. So these are the few like a few example of that the size difference should be 15 fold and you can see when the line width are becoming broader so as you can see we can monitor this ligand to protein ratio as we are increasing the ligand here free ligand it is a sharp line but like when binding becomes tighter and tighter you can see line becomes broader. Just looking at the line width even one can find it out STD is another experiment which tells about the binding property of the molecule. So to summarize this line width based analysis sharp line for ligand small molecule binding to protein it is a broad line. So narrow line, slow relaxation or fast tumbling and broad line because of the fast relaxation or slow tumbling for large protein. So looking at the line width one can find it out the binding probability or the binding capacity for a binder okay great. So two of the ligand based methods I discussed today to just give you an overview application of STD one can determine the KD that little bit more detail I am going to discuss in the coming class. So not only binders you can determine the KD association and dissociation constant K on and K operate and receptor can be of any type of protein either soluble or even membrane interview. So I am going to discuss this how you can use this for even the membrane integral protein to find it out the KD the binders using STD NMR and yeah the whole cell can be even used and at the minimum the need of protein is down like a 30 picomole you need really tiny amount of protein your ligand should be high but proteins should be very low to do STD NMR. So this has emerged as one of the powerful high throughput technique for drug discovery STD NMR of course I explained to you line widths. So in the next class we are going to use the advanced concept of STD NMR for determining the KD using like a whole cell lysate we can take it and how we can use this for say drug discovery the elucidation of binders. Another technique that we discussed briefly at the beginning of this lecture was a water log C. So we are going to also look at the water log C in the coming lecture and these are the ligand detected NMR techniques for understanding the protein ligand interaction then we can move to protein-protein interactions ok protein ligand interaction where we are going to look at the protein side not the ligand side protein side ok. So with this I would like to close today thank you very much see you in the next class. Thank you.