 So, good morning, welcome to today's lecture which is fifth lecture on protein ligand protein protein interactions. So, in last lectures actually we have looked at how we can use protein detected experiments to understand the interaction between protein protein and protein ligand. In this case if we are detecting protein most commonly used are like labelled protein, you can isotrifically labelled the protein using N15 or N15 C13 and then you can one can titrate with another protein or another molecule can be ligand and then look at the perturbation that is happening in each peak. So, that perturbation essentially reports whether interaction is happening, if interaction is happening where actually it is happening. So, not only the strength of interaction can be probed, kinetics can be probed and also the location. So, you can map those interacting residue on the structure and you can find it out where actually in the on the structure the ligand is interacting. Now, depending upon the rates with which the interactions happen they are classified into slow, fast and intermediate and I explained this with respect to NMR time scale what is called slow, what is called fast and what is intermediate and we can detect the binding cytrone protein as I mentioned. So, if something is slow that means you can simultaneously to see two peaks. So, one is for free protein another is for protein plus ligand and simultaneously seeing two peaks says that this is happening at slow time scale. So, that means one average PQC and intermediates generally you broad PQC. So, these are typical thumbs up rule of knowing whether the exchange is happening at slowest time scale, fast time scale or intermediate time scale and using all these information actually you can map on the protein side. So, here is my protein suppose and here some residue that are showing exchange or interaction. You can map those actually you can find it out where the interaction is happening. So, this is this binding site you can write it binding site on protein. Now, even you can use this information to create a docked model or a complex model of protein ligand. So, that is what we can learn we learned in the last lecture. Today I am going to go little more advance and discuss two of the techniques that basically proves the exchange that is happening at a microsecond time scale where KD is in micromolar range this we call as say typically intermediate time scale. So, what happens suppose you have studied the folding landscape, now suppose the protein is binding and populating another state which is not quite pronounced. So, like say protein is binding and remaining protein is binding and only populating 5 percent or 2 percent or 10 percent of these states and these are in constant exchange. So, now can we detect this 5 or 10 percent of bound state. So, actually say these are lowly populated say probably excited state lowly populated means like their quantity is less 2 percent 5 percent and excited state because they are somehow change their conformation from one conformation to another conformation and they are populated in low concentration. So, low concentration. So, can we detect these states right. So, can we detect these states using NMR experiments yes we can and that is what done using called CPMG experiments or relaxation dispersion experiment. So, we will be discussing today what actually is relaxation dispersion experiment briefly not very much detail because this requires quite elaborate explanation, but quite qualitatively I can tell you that how these experiments can be used to probe this lowly populated and excited state which populates only smaller fraction like 2 to 5 percent or even maximum up to 10 percent. So, how we can detect this. Now the question is that here we have a ground state or say energy minimized state. So, here is my landscape funnel and here are various states that populates. So, one of these is say one of the state and it is exchanging with another state which is also near native, but this is ligand bound state and this happening in only two state fashion. So, CPMG mostly deals with a two state exchange and two state exchange bound and free states. So, now not only it detects the K exchange rate, it also finds it out population of PA and PB. PA is a state which is say here the unliganded state, PB can be liganded state or excited state. So, the rate the like a kinetics the thermodynamics and also population it detects it. So, how it detects let us look at little bit more in detail. So, this is called CPMG experiment, CAR, PERSEL, MOBIUM, GIL experiment, relaxation dispersion experiment and basically this is used to characterize and quantify the binding process in the intermediate to slow exchange regime, intermediate to slow exchange regime using relaxation dispersion experiment. So, this is nothing but a Q2 experiment, we have run like launch the Q2 experiment or R2. So, R2 is transverse relaxation, so R2 has two component R2 intrinsic plus REX. This REX is telling about the exchange process that is happening and R2 is R2 intrinsic. So, when we measure R2, we essentially get both of these things, intrinsic transverse relaxation rate and the exchange rate given by REX. So, this REX has this phenomena of exchange can we use those experiments like a CPMG experiment to find it out what is the exchange rate happening. So, this very effectively probe the K exchange rate and as low as like 0.5 percent of minor populated state, often these states minor populated state is called excited state or many people call it dark state, invisible because you can imagine the 99%, 98% is populated by the ground state like that is what we hear and some states which is very low populated 0.5, 1%, 2%, so that is a dark state because the signal will be dominated by this ground state 98% fraction, but still NMR enables you to detect this 2% or 1% states which is excited state, but it is invisible state. So, how we can do that by doing REX experiment? So, quickly I will just try to explain you what essentially we do. So, here is my state A which in energy landscape here and here is my state B and they are in exchange, right. So, this is say 95%, 98% this 5%. Now, you have to do record this CPMG experiment at various like where you vary the distance between the pi pulses that I show you in the previous slide. So, here pi pulses if you see is 2 which gives you the R2 effective here and if you increase this you get R2 effective here and intensity decay is basically this intensity decay as you vary the frequency of CPMG pulses you can fit this and find it out the exchange rate, the fraction of like a population of A state, population of B state. So, for each residue using this HSQC based experiment you can do this relaxation this person experiment, fit the intensity and you can find it out not only the population like what is the fraction, the exchange rate, but also the probable structure of the alternative state. What I mean by say here is my major state and here is my minor state which is like say 2% only this is 98% so mostly what we do we get signal from here. But when we do this kind of CPMG experiment you can also get some information from this 2% state the 2% one and using this curve we can fit it out and find it out what is this other state, invisible state chemical shift. So, if you can figure it out the chemical shift of invisible state using that information essentially you can find the structure even the structure of the invisible states. So, that is what relaxation dispersion experiment enables you to finding it out Kx exchange rate between these two the population P a and P b of these two states and also the structure of the invisible state. So, what essentially we are doing is in this constant time we are changing the pipe pulse frequency and because of this the intensity decay is happened and intensity decay of R2 effective can be fitted it out to find it out all these parameters the exchange rate the populations and also the chemical shift of the invisible states which essentially tells you about the structure of the invisible state. So, how this actually happens? So, simply like a very beautiful work done by Louis K and his former colleague called Antonio Mittermeyer he is still working on these areas. So, essentially they explained it very, very like a nicely in a relatively easy manner giving an example of a runner and a walker. So, suppose a bunch of people are running a cross country race now there are a bunch of people who run fast and some people run slow. So, say they are running 5 kilometers. So, like they starts at t equal to 0 and certain time t equal to 1 hour there is somewhere and you see there is a large distribution, large distribution of distance between them what they cover. Who run faster they can quite easily reach to the goal and those who are really slower they will reach somewhere 2 kilometer, 3 kilometer. So, you have a large dispersion. So, here is what we are saying. So, here we started and they are running. So, they reach somewhere depending upon on the distance. So, here you have large dispersion. So, here is our effective large dispersion. Now you are say a referee and what you are doing? So, they run half an hour and you ask them you blow a whistle and ask them to return back. So, when they return back then you see that dispersion between them dispersion means like distance that they cover between them it will be slower like a it will be narrow and if you blow whistle many times the difference between the distance that covered will be minimum. So, that is what we have at the bottom. The dispersion is very minimal and when we have only one pipe pulse or two pipe pulse dispersion between them is very high. So, because of this dispersion measurement sorry dispersion measurement that we are doing two pipe pulse and many pipe pulse you see the R2 effective here is high and R2 effective here is low and essentially you fit this curve to find it out all those parameter that we are discussing. So, this is the minimalistic experiment explanation that I can give you how this relaxation dispersion experiment does. It is a really powerful experiment and it is very much used in understanding the enzyme kinetics, understanding the protein-protein-protein ligand interaction where the other state is populated really low as low as 5 percent or 0.5 percent. So, that is all about relaxation dispersion. Let us move to another important experiment called ZZ exchange. So, ZZ exchange essentially proves the slower process where the K exchange rate ranges from 0.1 to 10 of seconds. Suppose two states are really exchanging slow. So, say suppose protein one protein is going from monomer to dimer. So, it is a self-association protein-protein interaction self-association happening this is say monomer and this is dimer and this exchange between them is happening really slow order of say 0.1 second to 10 second. So, this is slower at the NMR time scale. Now what happens? You record this ZZ exchange experiment and what you do? You put them in Z magnetization and let them mix whatever we do in Noji experiment. Let the magnetization be mixed. So, to start with we are seeing a monomer peak and a dimer peak and if the exchange is happening between them as you increase this time 400 to 800 second you see you are starting this cross peaks. Now this cross peaks is telling that they are self-associating there is exchange between monomer and dimer and you can find it out the rates at which it is happening. So, here essentially if you look at we are increasing the time mixing time and because of this when they are mixing more you are getting a cross peaks. So, that is measuring the self-association or protein ligand it can be used for protein ligand interaction even the catalysis that is happening at slow similar concepts can be used to probe the cross like probe to the phenomena that is happening at the slow exchange ZZ exchange actually offers this. So, probably I covered you all the range the fast exchange intermediate exchange and slow exchange for how it happens and what it happens. So, if we understood this can we move ahead and just try to understand can we like a essentially can we get it more quantitative manner. So, in the Z exchange as I discussed it is a longitudinal magnetization created allowed to transfer from one state to another state or a measure bound state to unbound states and the here are the cross peaks that are appearing. So, population of K exchange between 2 can be determined by the intensity of these cross peaks of volume of the cross peaks with a different mixing time. So, intensity may change like this and you can find it out the K exchange that is what about ZZ exchange. Now, once we have the idea about the slow exchange, the intermediate exchange, the fast exchange can we get slightly more quantitative try to fix this parameter and do it and get some parameters that one can do it. So, how we can fit this NMR observed binding events. So, for getting a quantitative estimate we need to know something like we need to know what is the concentration of protein, starting concentration of protein which is P0. In what range this KD should come so you should have some idea from any orthogonal techniques like we discussed ITC, SPR or any other technique fluorescence based. So, what is typically order of the magnitude we are getting that will be good idea to have this. So, you should have an idea of the some KD and the concentration of protein should be of that order of the order of KD. The total ligand concentration say L0 should be one-tenth or ten times of KD depending upon what is there and one times of KD. So, if we do and we are doing a titration and probing this chemical shift suppose change. So, it slowly goes and after that certain time it is it saturates. What is saturates? The perturbation in the chemical shift as you increase the ligand concentration the CSP changes and saturates. So, you can if you know all these parameter what is the P0 the initial concentration of protein, initial concentration of ligand and the KD, KD that probably suppose we want to determine what we are seeing delta observed. So, how much actually we are seeing we can fit essentially these equations and we can find it out KD or if we know typically KD we can even predict how my observed chemical shift is going to come. So, essentially here delta observed is the what is the chemical shift that we are observing at a particular ligand protein concentration this is the maximum that we are observing here. So, we can if we are assuming simple two state happening two state exchange happening between free form to protein ligand form essentially we can feed all these parameters and find it out the KD of the binding event. So, let us see how we can do that I will give you some of the example. So, here like a binding event happening two exchange single site binding happening between the protein and ligand. So, typically suppose we take a protein concentration of 100 micromolar that is what I said KD if the KD is in micromolar range you can have the protein concentration of 2 in the same range so 100 micromolar. Now the maximum shift on saturation delta max can be say 1 ppm for a highly concentrated ligand and KD 0.1 millimolar of protein concentration same as the KD so protein concentration is same as the KD. So, depending upon how we ligand concentration we vary you can see a different saturation curve. So, here it is going and KD is typically of 1 now if we are saturated quite easily so ligand is saturated with ligand is saturating the protein KD is 0.001 and in different case one can simulate what is the KD with typical these parameters. So, that is single binding site the KD calculation with the shift change. Let me repeat again so typically you are taking 100 micromolar of protein and we are titrating with the ligand. So, here you can see we have taken millimolar of ligand 0.1 millimolar, 0.2 and our protein concentration was how much 100 micromolar right. So, that is what we are starting with 0.1 millimolar right yeah 10 to the power 0.01 So, 1000 micromolar is 1 millimolar so 0.1 millimolar of protein we are starting. So, when we put 1.1 millimolar if KD is 1 we are getting a straight line here it is increasing like this it will saturate somewhere going here. Now if our KD is slightly higher 0.1 we are getting saturation almost now reaching here and if KD is very strong like a very minimal in micromolar so here 10 to the power minus 3 micromolar so in millimolar case you will see that it is saturating very fast so depending upon the strength of the binding ligand protein concentration which choose we can find a different kind of curves that can be interpreted to find it out KD. Now another thing one can say here say ligand was fixed at 0.5 millimolar and protein concentration is being changed so depending upon what KD we have for different protein concentration you see a different kind of curve that is coming right. So that is what essentially you can find it out with the chemical shift change. So let us look at some of the example so here what I am showing you interaction between two protein one is called PDI protein disulfide isomerase which is interacting with a intrinsically disordered protein called alpha-synuclein this protein is involving neurodegeneration. So we are titrating this alpha-synuclein with PDI so first thing what we did we end 15 label this protein and PDI was unlabeled with no isotropic label we recorded HSCS spectrum of this protein and then we are titrating with PDI. So here we can see the nicely dispersed spectrum of the alpha-synuclein. You can see the like a although it is a narrow range but the peaks are very sharp and very round shape and now if you have such good resolution you can monitor even peak wise what is happening. So once you start titrating your protein here the other protein is 1 to 0.1 in the first case one alpha-synuclein 0.1 PDI you see some of the peaks already started shifting I have blown up picture here then when I am going to increase to 0.25 some more peaks here you can see start appearing sorry start shifting at even more 0.5 you can see lots of peaks now seems to be showing the chemical shift perturbation and one equivalent you can find lot more are showing the chemical shift perturbation. So here in the zoom picture you can see the shift is happening here and M5 again you see S9 you see L38. So many of such peaks are showing shift now that is fantastic. Now once you titrate you know how the protein ligand concentration is changing here L concentration you have and here delta delta you have a change in the chemical shift. So this is the thing that if you know the chemical shift change you can probably fit it and find it out how they are binding. So that is what one can do. The another thing if you notice what is happening here few of the peaks are showing decrease in the intensity if you look at closure two things are happening one shift in the resonance frequency or chemical shift perturbation the second thing happening is some of the peaks are showing decrease in the intensity. So that also can be plotted and what we found that here essentially some of the peaks that were showing decrease in intensity are coming from the N-terminus of the protein and some were from the C-terminus of the protein. So these from the N-terminus upon PDI titration of alpha-synuclein can be measured basically of a mutant and the wild type you can see there is some variations when you take a like a mutant essentially in the wild type you see lot more peaks are coming. So even changing one residue lots of variations you can see it. So these two NMR observable can be used for fitting and getting the KD. So now the chemical shift perturbation we can fix it here is the PDI concentration here is the protein concentration here is the ligand concentration and this is the shift in the chemical shift. So 3D plot we have made the peaks are shifting here and this shift essentially you can plot it plot to find it out KD. So one can find it out the KD of these residue specific KD of alpha-synuclein which is found to be in micromolar range. So for some it was 8 micromolar some for some it was 1, 26 and 48. So what we can infer here? Now KD you can find it out using the classical thermodynamic technique like ITC or SPR. SPR essentially gives you K on and K off rate which can be used to calculate the KD. ITC gives you delta G, delta H, T delta S and a stoichiometry you can even get the thermodynamic parameter. But what NMR is offering here? Not only the value the KD but also offering the residue specific KD which side of the protein is contributing more towards the binding which was not possible in ITC or SPR in single experiment. Yes you can mutate the protein create a different mutant and look at the relative importance of the one site C-terminus, N-terminus or midi site. But in a single experiment now NMR is offering you to find it out residue a specific KD of these bindings. Another example I am giving you where intermediate range regime binding was there. So this is the example coming from a sumo it is E2 interaction sumo is ubiquitin small ubiquitin related modifier it does the protein modification while binding to various enzymes. So one of the enzyme is called UBC9 that it binds and it forms a complexes. So when we did like titration what we see that upon increasing the concentration 1 to 0.5, 1 to 0.1, 1 to 0.2 lots of peaks are changing they are showing decrease in the intensity. Prominently in this region in this region and this region, but other regions were also participating. Now here the chemistry perturbation is not happening. So I cannot fit that data to find it out what is the KD. So but we use again orthogonal techniques and we find it out the KD is coming to be 4 micromolar. So you saw that here essentially in the previous slide we found that 8 micromolar there was a reasonable chemistry perturbation, but in this case it is happening at intermediate exchange when the two protein is binding, two globular proteins are binding the peak starts disappearing which is saying intermediate regime binding and which is substantiated by the KD and now intensity is started disappear. So the NMR time scale and its relation to binding is very crucial to understand that depends upon what is the KD, what is the K on it, what is the K operate, what is the ligand concentration. So sometimes it is possible that affinity can be in nanomal range, but the process happens in slow exchange. It is exchanging between the two peaks like for an example in this case you see here is a free ligand peak only one peak, but in the bound form it is shifting completely towards other state, but in between you see the other peaks starts appearing and one of the peaks start disappearing. So slowly this peaks disappear and this peaks started building. This is slow exchange process but the binding happens in the nanomal range. In another case one can see that here binding happens in fast exchange because you see continuously the peaks are shifting here. Binding happening in fast exchange but the affinity is in the micro molar range. So the protein-protein interaction which observed at protein comes in various forms depending upon what is the exchange rate and what is the affinity. So here fast exchange happening at a micro molar range. In the another case it was slow exchange happening in nanomal range. So that is what is protein-protein interactions. Let us summarize what we saw it today. Protein ligand interaction happens with different time scale and different strength, strength in terms of KD and time scale in terms of the exchange rate with respect to NMR time. And one can reliably fit the chemical shift data to find it out the KD. I show you some of the examples of interactions where you can look at the chemical shift perturbation or look at the disappearance of the peak to get the idea where basically these protein interacts. And you can using this information you can create a complex model. So here with this I am going to end now protein-protein and protein-ligand interactions. And in next week we are going to now see how this information can be exploited to understand the drug design and drug development using this protein-protein protein-ligand interaction. Can we come up with a molecule and then grow this molecule like in terms of chemical synthesis to make it effective or efficient drugs. So that is it going to be discussed that is what we are going to discuss in the next week. Thank you very much and looking forward to see you in the next class. Thank you.