 Welcome to today's class. So in the previous lectures we have seen how protein structure can be determined. Now here onwards we are going to look at some of the application of those structures that we determined. How we can understand the protein communication between protein, the interaction, the drug interaction, drug design and all those. So here onwards I will be taking you through all these important concepts of protein ligand protein-protein interactions and also followed by how we can use these to design a drug molecule. So protein essentially are highly diverse molecule and they offer a communication pattern. Actually they communicate with various molecules. It can be small molecule, it can be big molecule, it can be even electron, protons, all sorts of light it can interact with or some of the like a physically understandable molecules like a small molecules like drugs. Protein interacts with a drug, many of the drugs you can only use like asperin, ibuphen, some or other protein interacts with these molecules. It can even interacts with lipid, all sorts of membrane protein are in lipidic environment and interacts with a lipid and regulates many of the important functions. It interacts with a DNA and like one of the DNA packaging machine the you know nucleosome is a nucleic acid protein interactions or it can interacts with other molecules. So their interaction actually represents the stretch from atom over a small molecules such as it can interacts with a sugar, lipids or macromolecules. So this dynamic personality of proteins through which it interacts actually offers a very elegant tool to understand many of the biological functions where protein is involved. So what all essentially it does? So actually protein-protein interactions is a master regulator in cellular communication. How the cellular communication happens through protein signaling or protein-protein interactions. So they act as a glue to drive important phenomena in biology. For an example in receptor activation there is some protein-protein interaction signal translates on like a DNA replication. Some protein comes and interacts with a nucleic acid to start the DNA replication. Then invasion, it is a viral invasion, bacterial invasion, some pathogenesis is driven by protein-protein interaction. Therefore it becomes paramount of importance if you want to understand this like how cellular communication happens we need to understand the dynamics, thermodynamics, structural aspects of protein-protein and protein ligand interaction and that is what we are going to do mostly this week. So protein-protein interactions can come in various shape and size. The two proteins can find a shape complementarity where they can interact or even they can like the disorder protein can bind to a protein and takes order. So binding effect, binding can have a direct effect like when two protein interact they can directly interact and they can bind with each other and do its function like a signal transduction or it can have a elosteric effect. So direct effect how they do basically upon binding some type of conformational change happens and that conformational change lead to signal transduction. So that is a direct effect, it can have a elosteric effect, elosteric effect in a simple term it binds to a site, elosteric site and the effect of this binding is seen somewhere else. So that is a like here the region which is regulated by elosteric binding happening somewhere else the effect is transduced somewhere else. So that is a elosteric effect. So protein-protein interaction or protein ligand interaction in general can have a direct effect, bind somewhere changes the conformation and this conformational change actually initiates the cascading effect and that is how the signal gets transferred or it can have a elosteric effect and that is how the signal goes from one place to other place. So if this is so important protein-protein interaction, protein ligand interaction if it is so important because it tells about the cellular communication, cellular regulation can we understand this quantitatively. Quantitatively I mean like if two proteins or one protein, one ligand is interacting what is the stoichiometry in which ratio they are interacting? What is the stoichiometry, how they are like what is the kinetics of their interactions, what is the forces involved, energetics or thermodynamics involved in their interactions. So these are the some of the critical parameter that we need to understand the stoichiometry that means I mean to say ratio of their interaction, the kinetics, the rate with which they are interacting, k on rate, the rate with which they bind, k off rate, the rate with which they goes, so k on k off rate can be understood. What is the thermodynamic parameter like what is the enthalpy involved in this, what is the free energy, what is the entropy can we understand all those thermodynamic parameter quantitatively, stoichiometry, kinetics and thermodynamics. So for doing that there are various biophysical methods all sorts of like a biophysical methods are there which actually which which offers some of these where we can understand the protein-protein interaction, protein-reaction quantitatively, we can understand the magnitude in affinity like with the strength with which they are interacting, they can even monitor the kinetics of interaction, the rate with which they are interacting or like a what is the lifetime of the complex form like how long they stay in this complex form that also they offers to understand. However, we look at some of these methods biophysical method they are used, but however NMR spectroscopy is a method of choice that basically address the protein ligand interaction in a very elegant way that also we are going to look at. So let us start with some of the some of the basic concepts of protein ligand interaction. Suppose a protein has one binding site where ligand is binding, so we can write a simple reaction like protein binding to ligand and forming a complex and this PL is in equilibrium with the free protein free ligand site. So assumption is protein has only one binding site and so we can write this equation. So the ratio between the concentration of the molecule in the free form free state of protein and free of state of ligand and the concentration of the complex PL, if we get this concentration we can get the equilibrium constant. So we can write it k equilibrium how much protein is in the free form, how much protein is in the complex form and that is how we can get the k equilibrium on this reaction, so basic chemistry. So then we can get a rate, the rate with which they are associating k association and rate is more inverse then we can have a also k dissociation the rate which they are essentially dissociating, so like they are going back to the protein ligand form. So we can know the rate of k on or k of k on means k association k dissociation and we can determine the dissociation constant of a protein which can be seen like this. So kd is a simple, simply the free ligand concentration at which 50% of protein population is bound to the ligand. So that is a kd, so like you have heard about various kd we will be explaining those kd little more in detail but actually it is just it is a term that determines the ligand concentration at which 50% protein is bound in the ligand form. So looking at this simple reaction we can understand something about rate of association and rate of dissociation and this dissociation constant. Now these strength of the binding can vary depending upon what sorts of interaction is happening and they can vary like order of magnitudes. It can be nanomolar to millimolar. Nanomolar kd means very strong binding, ultra strong binding is nanomolar. We can have a ultra weak binding which is like a more than millimolar and we can have an intermediate which comes somewhere in the micromolar range. So we can have all sorts of kd. Most of the biological phenomena occurs where the kd is in micromolar range like they are transiently binding with a micromolar strength and they goes off. So these essentially forms lots of cellular communication. So basically NMR spectroscopy is capable of providing quantitative information of protein ligand interactions affinity which is even lower than the micromolar range. Actually essentially NMR can report about the kd ranging from nanomolar to millimolar range. But before we go to the details of that let us go little bit more detail that what actually the complex formation landscape for protein-protein interaction is needed. So to start with we can determine the protein structure using various NOE based experiment or RDC based experiment we can even get the parameters like of the diffusion of the molecules like hydrodynamic radii and the kd and these are sensitive on 1D and 2D. So let us look at complex landscape of protein interactions. So when two molecules are in solution they diffuse all the time like they are tumbling they are diffusing they are doing transglacial diffusion even rotational diffusion they come and encounter with each other. So say protein a big protein molecule we have here and a small ligand they are diffusing and then they are coming and colliding right. So they occasionally encounter each other and depending upon how precise the orientation is. So this is of course binding site and here is ligand so through searching the appropriate binding site sometimes it happens that they find the precise orientation and then two molecules form something called encounter complex right. So here is say binding site and here is my ligand. So it searches all the possible site and finally when it finds the right site it comes and binds. So that is we can call it let us say correct binding happening. So when they form a precise orientation that will be called encounter complex and encounter complex is needed for the proper interactions. So the energy landscape we can say the two proteins here quite disorder protein and here little order protein they are in unbound form they are one energy landscape which is like a zero and when they form a encounter complex when they are coming closer but not so in precise orientation they form a encounter complex you see the energy is dropping down. So that means they are becoming more stable and then they finally adjust to bind to the appropriate binding site that will be called allowing encounter complex. So you can see energy further drops down and now that forms the stable protein ligand or protein protein complexes. So first thing we learn they have to come closer by diffusion then they have to find a proper orientation and finally when they fit each other that is called aligned encounter complex the energy is down in the bound form this is the stable state that we have formed. Now when this encounter complex forms it can have various way of searching it is a right confirmation. So two or three already we have learned right in the previous cases it is called induced fit or conformational selection these are the two known concepts in the protein ligand or protein-protein interaction case. So what happens that conformational selection says that there are various multiple confirmation at the protein energy landscape available ligand starts searching the right kind of confirmation and so it searches between all equally probable energy states and one of the energy state which fits better it binds and lowers down the energy that is called conformational selection. In induced fit what happens that the confirmation of either ligand or protein changes and basically it finds the right way to energy stabilize so it can change its confirmation from here to here and that is how energy state is stabilized. Now there are various now recently some of the more states has been discussed which is called conformational restriction. So like in the previous slide we saw that the two proteins in which there was one protein which was quite disorder and now it binds and takes some more order this kind of the conformational selection we can say conformational restriction is happening or it can even happen to accommodate a protein or a ligand the other receptor proteins extended its confirmation that we will call is a conformational extension. So you can see here the like here is suppose ligand and blue one is a protein now confirmation is getting restricted here you can see confirmation of the red is getting extended. So this is a conformational extension confirmation means like a more equal probable energy states are coming and that is a conformational extension or it can have a mixed of some conformational restriction some conformational extension or it can even shift the confirmation that is called induced fit. So one of these two methods basically protein interacts with each other so if you look at carefully what is happening here two things are happening shift in the energetics of protein protein or protein ligand interaction. So that means the thermodynamics is involved here second thing what is happening here is a structural change happening because protein is changing its confirmation either restricting or extending or like a induced fit also again confirmation shift is happening. So structure plus thermodynamics both things are changing. So if we want to understand protein-protein-protein ligand interaction essentially we need to get hold of all those changes that is happening. Now most of the biophysical or structural techniques that are there to understand protein ligand interaction we can classify them that they fall in two groups either they measure the thermodynamic or kinetics of interactions. So they measure the thermodynamics or kinetics of interactions some of them we can classify as isothermal titration calorimetry which measures the essentially thermodynamics the surface plasma resonance again that measures the K on and K off rate it is a measures thermodynamics. Dynamic light scattering essentially it measures the how shape and size of the molecule changes briefly I am going to discuss all these and then there are techniques which illustrate structure that may happen upon interactions. So those again we are briefly going to study the thermodynamic and structural techniques but and then we will come why NMR can be used to study protein-protein interaction. So essentially let us focus on the thermodynamic parameters that are there one of the prominent one is isothermal titration calorimetry. What happens here it is isothermal means same therm temperature titration we are titrating it and calorimetry because it measures the heat. So same like we are maintaining the same T like therm isotherm we are titrating two things and measuring the heat that is why it is called isothermal titration calorimetry. So what essentially we are doing we have two cell one is called sample cell another is called a reference cell. They are maintained at at the they are maintained at isotherm with some thermocouple and they are in adiabatic jacket so that no heat transfer happens with surrounding. Now here is a reference cell that has a feedback loop, maintenance temperature and here is my sample and here is my ligand sample ligand we are titrating so we are injecting each time here so you can see injector and here sample cell. Now this is my protein shown in red and blue and orange and yellow is my ligand so we are each time we are adding some ligand. Upon addition of ligand heat change happens then again it brought back because we have a reference cell and sample cell. So brought back to the isotherm again we add and then heat change happens. So because of this heat change happening we are measuring here dq by dt change in the heat with time. So that is what we are measuring change in the heat with time microcal per second each addition of ligand there is a heat change. Now what happens that after this after sometimes it gets saturated and you see there is no further heat change. Now what we will do we will fit this equation which is here and from fitting up this equation essentially we get various thermodynamic parameter delta H the change in enthalpy change in entropy delta G and change in astratometry or N. So how many molecules binds to one protein molecule that is delta N what is the free energy change what is change in entropy and what is change in enthalpy. So this isothermal titration calorimetry is a wonderful technique to understand the thermodynamics of the protein ligand protein-protein interaction. The other one which is used commonly is called surface plasmon resonance it also measures the thermodynamics. So it is like here you immobilized your receptor on the sensor chip and here is a prism you have a light source optical detection unit and whatever like upon binding it forms plasmon then refractive index is changed and that essentially gives you the rate of association so that refractive index change is measured in terms of response unit and like here you can see the angle changes upon binding when you flow some ligand. So upon binding the flow channel has so upon binding so here is a receptor here is my ligand coming and binding upon binding some response unit change that you plotted. So what happens when it starts binding it shows a curve which is called association curve so K on association curve and then finally you wash with buffer then it dissociates and then you regenerate your chip so that the next set of experiment can be done. So here is analyte injection you do it associates then it saturates you can see here saturation happening and then you dissociate it. So with time we can measure the K on rate, K off rate and that again gives you the KD. Precisely surface plasmon resonance give you the on rate what is the rate with which ligand bind to a protein what is the rate with its gate dissociate and then finally you can calculate the KD. So very important it measures the K on rate and K off rate. The third one that we talked is essentially dynamic light scattering that also gives some idea of protein ligand protein-protein interactions so essentially this measures the how the molecule fluctuate in the solution. So if you have a small particle that is fluctuating you can plot a correlation function and if it is a small particle tumbles very fast so you can have a here correlation function that shows the decay is happening fast then large molecule slowly tumbling you can see the intensity of this large molecule is like a quite broader. So here you can see when it form a complex you have a different kind of correlation function and you can plot it to get how the shape upon interactions has changed, how the size molecule has become bigger so these are the three techniques essentially this does not give to the thermodynamics but gives idea about the change that happening and this is again low resolution technique. So now coming back to upon interaction some of the high resolution techniques that gives you the complex structure the static three dimensional picture of protein protein or protein ligand complexes one of them leading technique that is used is essentially x-ray crystallography. So what happens that take a protein and ligand or two proteins you crystallize them diffracted and using the electron density map you can get the three dimensional structure of protein and protein interaction. The another one not so precise but now it is quite used because it is sensitivity is called EPR again you have to have a paramagnetic tag add and then one can get the protein complex interactions how the paramagnetic tag interacts with the other partner one can get the structure of a protein. The third one is small angle x-ray scattering right so here essentially when the it is kind of a x-ray scattering but does not give high resolution atomic resolution structure gives a overall shape and size of a molecule. So here you have to like again the complex which forms bigger will give a different kind of a scattering pattern so you know that now complex form hazard. The recent phenomena of cryoem that is excellent tool for understanding the bicar complexes is coming more and more profoundly which understand the three dimensional structure of a protein ligand or protein complexes. Another very important technique called analytical ultracentrifugation essentially that also says like that also reports upon complex formation how the molecule sediment. So you can measure the sedimentation coefficient and that also says that the complex formation happens. So essentially the sedimentation coefficient is lower when it is in the free state and when it is in bound state becomes higher. So essentially you get the shape and size of a molecule that can be complementary to dynamic light scattering. So AOC cryoem, SAX, EPR and x-ray crystallography gives you the static three dimensional structure picture. Another important techniques where you can even get the dynamic mode of association is called Froster resonance energy transfer a fluorescence based technique where you have a two fluoropore attached on two molecules can be ligand and protein and how they come closer and get associated you can measure the distance between these two fluoropore using this fluorescence technique called Frit and then measure the dynamic mode of association even the mode of complex formation. So these all techniques thermodynamic and the structural mode of interactions can be used. Now the question remains then what NMR can offer you can NMR offer simultaneously both of these techniques these aspects structural as well as the thermodynamic and kinetic mode of. So we know that one can understand the structure of protein already we have seen that can be determined using NOE based restraints the dipolar interaction the distance restraints helps us to get the structure of a molecule and we can determine the structure. Once the structure is there the static or complex structure we can get the structural details for there probably you need a labeled protein and you can have a unlabeled ligand. So using these we can determine the structure what you need a spectrometer and most of the time protein labeling is needed. We can determine the structural details at atomic resolution and also we can measure the kinetics of binding thermodynamics of binding. So here onwards we are going to delve deeper into how we can measure the thermodynamics and kinetics simultaneously along with the structure of a protein ligand or protein-protein interactions. So what you need for doing NMR essentially the protein concentration which should be more or like a 500 micro molar or more you need a ligand which would be like a typical depending upon binding constant you need to have a more than 0 micro molar some micro molar concentration you need. The stability for these interaction proteins should be stable for 24 hours or so and temperature is also important many proteins are stable in this range so temperature is also important what temperature we choose but one thing one caution is there the exchange rate depends upon the temperature. So if you go at higher temperature the on-off rate can be different so you need to choose an appropriate temperature we want to understand the complex formation. The pH is very important pH should be less than 7 and it should offer the good protein stability preferably less than 7 because MI proton does not exchange and you can detect all the MI protons. So pH you have to choose for doing NMR experiment which should be less than 7 which buffer a buffer which does not interfere with the protein signal. Many of the buffer like a mesh buffer and all those has lots of proton in itself that overcrowds the protein spectrum so we should avoid those kind of buffer phosphate buffer is the best buffer to choose but actually it depends upon where your protein is happy and stable. So buffer has to be selectively chosen and the typical volume for doing this experiment should be 500 micro molar. If we have all these we are ready to go for protein ligand interaction and that is where we are going to start in the next class how we are going to now use NMR spectroscopy to study the protein ligand interaction and getting the thermodynamic kinetic parameter from it. Structural part we have already seen but wherever needed I will come back and explain that. Thank you very much and looking forward to see you in the next class thank you.