 So good morning, this week we are discussing protein ligand and protein-protein interactions. Today we have a fourth lecture. So in the last lecture we discussed various techniques that mostly detected the small molecule ligand in this protein-protein interactions. Some of the technique that we discussed like STDENMR, saturation transfer difference, water loxie looking at the line width and transfer anyway, how these techniques can probe the ligand side and once we find the epitopes we can even create a docked model or complex model where because we know from where the ligand like what are the sites on ligand that interacts with the protein. So those can be used as a restraint to create a complex model. Now let us go into little more detail and today we will be discussing how you can look at the protein side. So the other partner which is a bigger molecule protein how we can look at this and that is what last time we summarized a ligand based NMR methods for screening experiment to determine in a qualitative manner STDNMR. We can map the ligand like a ligand epitope mapping and then we can use even if we do multiple concentration like what we discussed is called STD amplification factor we can do that and we can find it out the dissociation constant in a quantitative manner. Then we also discussed saturation transfer double difference for whole cell binding. So we ended up here. Now today we were going to look at the other side that is protein based method. So we can classify this ligand protein interaction into two ligand based method again it relies on knowledge of property of ligand small molecule typically then we can look at the chemical shift perturbation the relaxation that we already discussed and change in their resonance position and also how they are diffusing so diffusion coefficient upon binding the small molecule will diffuse differently than whenever it is free. So all those we had already finished it so all these are done. Now protein based method basically we are now looking at a bigger molecule protein right. So we need to have a knowledge of protein how what is the structure of this what is the function of this protein. So structural functional aspects of protein should be known when we are using this for a protein based method. Now actually the deterministic signal that comes from protein is chemical shift change change in the chemical shift so we can do SAR by NMR monitoring how chemical shift is changing and typically for doing this 2D heteronuclear single quantum coherence is used 2D heteronuclear single quantum coherence is used right where each peak gives an idea of one of the correlation like if you are putting it 15n and proton correlation so that means each peak which is here is reporting about one amino acid and the changes that happens in these amino acids essentially can be captured when we do protein based NMR methods. Right so what we can look at a protein detected experiments so you need to first stably like a label this with a stable isotope and here our ligand can be unlabeled. So protein has to be labeled because now we are looking at the protein and since protein molecules are big so to get a resolution to understand more in details because now we want to go and go to get residue specific information. So we need to enhance the resolution by isotopically label these proteins however our ligand can be unlabeled. So what we are going to monitor here is a chemical shift. Now chemical shift of a protein actually may change upon binding or intensity may change upon binding so one of these parameter we are going to monitor. So the change in the chemical shift change that the chemical environment of that particular nuclei changing and that basically can report about what is the on rate of binding, what is the off rate of binding, what is the KX exchange chemical exchange phenomena and using that actually one can get the KD of the binding right ligand protein binding. Then we can even find it out the binding site on the structure where from where the chemical shift is changing what are the residue that are engaged in this interactions that is going to tell us about binding site mapping we are going to discuss this in detail. Then the other parameter that we can monitor so if you remember in the last class I just on the basis of one day I showed that here is one parameter called chemical shift another parameter called intensity and third parameter is called say lambda line width these three are easily determined like we can determine these parameters easily. So chemical shift tells about the chemical environment, intensity tells about how many like a what is the population that is being participated in these. So intensity also can tell you about binding site mapping on the structure and using this of course one can get it K on, K off, K exchange and KD. The third parameter is line width right so line width depends upon T2 the transfer relaxation rate and if the molecule becomes bigger and bigger your line width changes and that also tells about this changes so for a unleganted you have a sharp line once it binds you go to a broad line so that generally the it may go depending upon how binding is happening you go to go and get the broad line and that also tells about the binding phenomena. Then we will be also discussing some of these other techniques like a CPMG techniques that essentially is used for determining the lowly populated state in the protein ligand interaction or even protein conformational change and ZZ exchange we are going to discuss both of these. So essentially depending upon what we want to probe so CPMG actually probes the exchange between two states and if they are slowly exchanging then ZZ exchange can be probed. So briefly we are going to discuss these two techniques in coming slide. So let us start from the beginning so as we said if we are looking at the protein we need to have some structure function information of a protein and just to recapitulate whatever we have done recap of the steps involving protein structure determination by NMR so essentially what we are doing we are going from primary sequence to three dimensional structure and what are the steps involving doing this first you have to prepare the sample. Now our sample has to be isotropically labeled so you feed your bacteria with ammonium chloride and carbon 13 glucose and or like you can be singly labelled only ammonium chloride and or ammonium sulphate or for doubly labelled your glucose also has to be 13 C labelled. So once we prepare the sample we can go and collect the data multidimensional data. So multidimensional data will be collected now since this is our said data coming in the background also have a data so we collected data. Next step to identify what is this peak what is this peak what is this peak so all these peaks identification is called resonance assignment so you have to analyze this data. Now we have a name for this these peaks it can be some L, M, N whatever amino acids so and for structure determination you have to generate restraints so distance restraints angular restraints and all those you have to generate which we have already discussed and then you calculate the structure three dimensional structure using these restraints angular restraints the bond vector restraints and the distance restraints. And finally we incorporate all these restraints for a structure calculation we determine the structure and finally we validate this structure what is the quality of the structure. So once we have a structure we are all set to understand how this protein is interacting with another protein or another ligand and that we can now we are going to use this information which we have captured here during the structure determination of the protein. So just to summarize suppose I have a protein which we are working in our lab say SKP1 S phase kinase protein some protein which is involving ubiquitination so it is one of the component in E3 ligates so do not go in technical details so first you have to purify this protein right. So you have to be getting the exact molecular weight so you can do either SDS phase you should have a single band of these proteins then you can validate using Maldi techniques so you are getting the exact molecular weight this is m by 2 so you are you are sure that my protein is absolutely pure and this protein sample that we are preparing is at least n15 level. We also determine the secondary structure using one of the low-resolution structural technique like circular dichroism so one can see here it is showing helical characteristic and you can see here lots of helices are there so now this corroborates that the structure which should be captured is showing helical. Then we recorded the HSQC spectrum using various sets of 3D experiments that you have already done one can assign each of these peaks so you each peaks has a name right so this is say V25 this is K113 this is say A52 so now we have now once you do titration experiment or the binding experiment the any perturbation you see here can be captured and that perturbation is essentially used to understand protein ligand or protein protein interactions that is how we are going to do now. So suppose this my favorite protein SKP1 is binding to its partner called SKP2 and binding site is here so what do we expect right from theoretical knowledge so this blue is SKP1 and this the orange color is SKP2 and binding site here is shown indeed so suppose we are studying this protein right we are doing the titration of experiment. So we assume we are saying that there will be some change in the chemical shift so to start with what we are doing we are taking the labeled SKP1 we are monitoring on SKP1 and then we are taking unlabeled SKP2 we are titrating it and seeing what is changing so suppose change is happening so I have taken here two residue here in this plot and here also two residue if you see one residue is very less changing something is happening that we are going to discuss little more detail so intensity is changing but here you can see it is a chemical shift is changing in this case also this guy is not changing A6 and say some name like A6 it can be from other protein so some amino acid is not changing and here one amino acid is changing very nicely if you look at if we are titrating it adding more unlabeled SKP2 peaks changes so now change in the peak at least is telling that some interaction is happening right some interaction is happening and now that is what we are going to monitor so chemical shift is absorbed for the various species and those are the powerful probe to understand the binding interaction. So we have taken the labeled one part not unlabeled another part not we are titrating it adding and we are maintaining some structure material for each titration we are monitoring what is happening in each peak and a peak wise manner we are going to assign and understand the point of our interaction on the protein side great. So as we saw there are there are certain phenomena happening peak is shifting or peak can appear appear at a different position or it can disappear so now appearance or disappearance of this individual peak that means change in the intensity or line width can vary and that needs to be quantified for finding it out actual binding effect and that depends upon several property right several property of these interactions so let us take it some of the parameter which will help us and understanding the protein-protein-protein ligand interaction so suppose we have a free protein which we define as a P it has concentration of P now or a characteristic P and a free ligand which is L and a complex which is forming PL so for a free protein there is a chemical shift right which is omega P and when you remember what we are looking at only protein ligand is unlabeled so when protein forms a complex which is PL now its chemical shift is changing from omega P to omega PL right so the change in the chemical shift that we are monitoring is delta omega that is happening because of this interactions so we can say omega P minus omega L so to make it more understandable here is my omega P and it has shifted and made it something like omega PL so this difference that we are seeing omega P minus omega PL is delta omega so change in the chemical shift of this protein which is labeled is delta P okay so upon this interaction we are getting this parameter now the appearance of different species of protein in the NMR actually varies it also depends upon what is the concentration we are choosing of various species protein to ligand test stoichiometry it depends upon what is the rate with which they are associating what is the rate which we are they are dissociating so Kd is one of the important and what is the exchange between them so how these two like two protein bound and free form is exchanging between them so K exchange in next slide I am going to or next couple of slide I am going to discuss in little more detail so let us define this K exchange parameter K exchange parameter is K on into ratio of ligand and plus K off so one can determine subpopulation of free protein one can get it K off divided by K on into ligand concentration plus K off and bound conformation can be P like a population of PL K on ligand concentration divided by K on L plus K off so if we know the ligand concentration if we know the rate and population we can find it out what is the free population what is the bound population so by getting these chemical shift and ligand concentration we can also get the population which is bound form which is in free form and various thermodynamic parameter so you can see now we are now going little more detail in a quantitative manner of binding in terms of thermodynamics in terms of kinetics. So as I have mentioned one of the parameter that we were discussing is Kx so chemical exchange this is called so two or more state right during the time of recording of NMR how they are changing that is called exchange phenomena suppose this is my protein cartoon we have made and this protein is changing its conformation right here it is a change in the conformation. So the rate with which the exchange between one conformation to another conformation when we are recording the NMR spectrum that is called chemical exchange. So typically on the NMR time scale this K exchange may vary it can be fast it can be slow or it can be intermediate so depending upon what is the magnitude of Kx. So chemical exchange typically can be slow if the two states are slowly exchanging with each other what is the slow with respect to NMR time scale again I am going to explain you what is the NMR time scale because we are recording the spectrum so when they are exchanging slowly so we can probably get the two peaks one coming from state A say state A and one coming from state B if they are exchanging very slowly the another phenomena can be intermediate time scale it is neither fast nor slow so like you see fan if fan is rotating very slow very very slow you can see all three wings right if it is with some speed they are like they are with some speed they are spinning where your eye cannot resolve it but you see and averaged right average state of these that we can call is intermediate states so where the contribution from A and B are so merged that we are unable to distinguish and then there is a fast exchange with respect to NMR time scale so then you see an average state like if fan is spinning very fast you see an average value of these three wings or average phenomena of these three wings so that is a fast exchange so at the NMR time scale the exchange between the two states can be slow fast or intermediate and that based on the magnitude of Kx right so what is the NMR time scale and that that we are talking and exchange rates so when we are probing and say protein ligand interaction using a standard either one-dimensional proton NMR or 2D NMR spectrum that is what we are going to do so for labeled protein we typically decode this HSQC but you can use also 1D NMR that we had discussed earlier right so what is the lifetime of say bound state and free state how these say bound state or P state and PL state how they are exchanging and so the lifetime of bound state and T free state is tau and then it depends how accurately we can determine the resonance frequency of this P state and PL state respectively so that is given by the lifetime of these states and that is say delta omega so the change in like a difference in the resonance frequency of omega P minus omega PL is our delta omega and that is correlated with this Planck's constant with a lifetime of these two states how what is the lifetime of bound state or free state so delta omega is the difference in the resonance frequency for these two peaks so K exchange that is per second will be given by the lifetime of this one by tau and that will be respect to this delta omega that is in radiant per second so if the lifetime of two states is very short very short then the difference in the resonance frequency cannot be measured right so that is that is they will collapse to a measured signal if the lifetime of these two states is very short so like they are so short they are exchanging very fast so you cannot measure it at the NMR time scale it will be unmeasurable you get a collapsed state but if they are slowly exchanging the lifetime is quite long you can measure it right so that is what happens in slow intermediate and fast exchange so when an exchange rate is much slower than the observed difference in the resonance frequency the exchange rate is is said to be slow on the NMR time scale right on the NMR time scale so here is a protein and here is elegant right so they are slowly exchanging so kx one can have which is 1 per second to 10 per second you can see in slow exchange depending upon what is the kdk of rate and all those we are seeing two peaks clearly two peaks one peak coming from the free protein one peak coming from the protein in complex with elegant two peaks we are getting so our k exchange is much slower than the delta omega change in the difference in the chemical shift of these two and even in 2d you can measure so we are getting clearly two peak one coming from the free protein one coming from the protein complexes which is here okay. So now this is called this slow exchange where kx is much less than delta omega the difference in the chemical shift of these two states. The another one which is called say intermediate time scale regime where our kx exchange will be roughly equal to the change difference in the chemical shift of P and L. So here suppose this is for P and this is for L so you can see now lines are getting broader and exchange rate between this so here line is going down here lines are coming up here also you see the lines are getting broader and lines shifting so depending upon what is the even in the intermediate regime what is the exchange between these two you see peaks is slowly shifting and disappearing that is the intermediate time scale. So as we were giving analogy of fan if it is speed spins so that we cannot differentiate between these two states our eye cannot resolve it that is what is the intermediate state k exchange. So here the difference in the frequency will be equal to kx kx of the of two states. Now coming to the first exchange this is the regime where our k exchange the exchange between the these two states omega p and omega pL is very fast happening very fast happening so k exchange is much faster than delta omega of these two so these two states are closer but peaks gradually shift to a new position and we have a average value that we see so this is fast exchange happening very fast. So these two concepts or these few concepts of slow exchange intermediate exchange and fast exchange are measured in terms of what is the difference in the resonance frequency of these two states at the NMR time scale with respect to NMR time scale and that is basically used in the NMR. So here one can see now in the fast exchange regime our peak is moving from one position to another position great. So now coming back to how we can use this so suppose in fast exchange regime the chemical shift in the either dimension either of two dimensions proton and nitrogen can shift either proton can shift from one position to another position nitrogen can shift to one position to another position so in that case one has to take the change in the chemical shift combination. Now how do you normalize because now proton chemical shift varies from 0 to 10 and this can vary about 30 percent in protein like 100 to 130 so you have a normalization factor that comes from the gyromagnetic ratio you can measure it delta 8 combined will be change in the chemical shift frequency of proton square plus 0.1 which is coming from the ratio of gyromagnetic gyromagnetic ratio 0.1 multiplied with the change in the resonance frequency of nitrogen just square it take under root that is a combination you can see fast exchange since proton and here proton and nitrogen both axes are moving so in those case you can find it out how much chemical shift is changing great. So we find it out or you just show you one example of non-covalent binding of two proteins so here in my lab one of my PhD student titrated N15 level sumo with its binding partner called E2 and he found some of the peaks were shifting little bit some of the peaks were disappearing so he quantified it and looking at these values since peaks were disappearing more so we are knowing that this is happening at the intermediate time scale it is not a fast time scale to substantiate that he did also ITC experiment that I explained you in the previous class this is a thermodynamic parameter experiment that measures the various thermodynamic parameter and one can see here when we titrate sumo with its binding partner called E2 we are getting a typical KD of 2.5 micro molar and looking at this peak disappearance few of the peaks you can see here either shifting or disappearance we are knowing that this is happening at intermediate time scale. So these two techniques are corroborating very well KD at a lower micro molar range and peak disappearance happening in the NMR one can say that the exchange between the bound form and a complex form of N15 level sumo was at intermediate time scale. So he found it out not only the position where it interacts the order of magnitude of the interaction also one can find it out. Now another example I am showing you here is another protein which interacts with one of the metal ion so we titrated with metal ion and what you are seeing here few of the peaks just zoom it here like here we have A119 you can see nicely the peaks are shifting here 120 peaks are shifting here. So these peaks were shifting and they are telling that these are the residue that are involved in the interactions. So you can map those interacting site on this structure now you know where this metal is binding. So not only you got the thermodynamic parameter by fitting it which I am going to explain in the next class how you fit this change in the chemical shift to get an idea of exact binding constant but also we got the location where actually it binds you can map those on the structure and find it out exactly the binding site that is what NMR offers you not only the position of the binding but also it also offers the thermodynamic parameter here qualitatively I told you what will be the order of magnitude next class I am going to explain in a more quantitative manner how we can use these to find it out what is happening. So there are like that I will stop it here and in the next class we are going to discuss to advance technique which is used for understanding the protein-protein interaction is called CPMG and ZZ exchange that captures essentially the intermediate scale exchange like a micro molar exchange and a slow exchange that we are going to discuss it and then looking at some of the quantitative aspects of interactions these interactions how we can determine the parameter. So hope to see you in the next class thank you very much for your attention thank you.