 So, we continue with the discussion of aggregates, how to use NMR to understand the process of self-association. And here you have an example of the same protein GD what was saw earlier and here you have a assembled state, so an aggregated state here and our challenge is to identify this. Each of these molecule has a certain structure and they have assembled to form a large aggregate. Now what we should do well one can do you can dissociate this into the individual monomers, break the associated state into individual monomeric states and when you make this monomer of course you will see a beautiful NMR spectrum because each one of them is a small molecule now. So, and that will give you all the peaks, all the expected peaks you will get here in this protein. Now you assign all of these using the standard methods which I have discussed earlier you assign the individual peaks. Now what you do is you start reconstituting this, so slowly change the conditions, slowly change the conditions and then monitor the HSQC spectrum as you are going through the process of association. See therefore a few of those are indicated here, from here you go to this then you go to this and finally when you reach here you come back to this situation where you have only small number of peaks. So in this entire process you can monitor the changes in the chemical shifts or the line widths of the individual peaks to tell you which peaks are associating, which are received are associating in the sequential manner. So that is the strategy, so that is how we can identify. Of course when you induce this initially this is all unfolded now, when you dissociate it they become unfolded but when you start the reconstitution process they will also start folding. When there is it will start folding of course there will be small chemical shift changes as well. So therefore you monitor the changes in the line widths and the chemical shifts to understand about the process of the self-association. Now here is an illustration of that, so you have the GED is dissociated using DMSO the dimethylsulfoxide. In presence of dimethylsulfoxide the assembly is completely dissociated. You will see all the peaks here and beautifully you can analyze all of these ones there. Now you start decreasing the DMSO concentration means these are all separate samples, you will have to prepare separate samples. So this is prepared in 100% DMSO, this is in 90% DMSO, this is 85% DMSO and so on so forth. When you have 90% DMSO you do not see as many peaks as are seen here, the few peaks are seen and many peaks have disappeared. Now you monitor those which are the ones which are changed, come to 85%, more peaks have disappeared, 80%, even more have disappeared, 70% more have disappeared and in 50% almost it is like the major of the original associated state. So you have stopped here, beyond that it does not help anymore because you already reach that completely associated state here. Now you analyze this, when you analyze this what you get here, this is what is shown here. This portion of the protein is the end term which is what you actually see here. This remains flexible, this remains flexible even in the associated state, this is what you saw in the previous example also and this remains associated you will see these peaks and these ones which are color coded here, these ones are happening in, disappearing in a sequential manner, stepwise they actually disappear as you are decreasing the DMSO concentration. This is and what are the changes that are happening in the protein with regard to the structure and with regard to the relaxation properties, that is shown here. So you see here, this is the secondary structural propensities measured using the delta C alpha. So with the delta C alpha, this is the secondary shift what we are measuring here and here you see that only a few residues, they are mostly they are down here and here it is everything in the same direction. A few of those are quite above and these are the where the helical propensities are there. This is indicating helical propensities, a few stretches have a helical propensity there. Now you decrease, helical propensity increases. So this area also gets the helical propensity, this gets the helical propensity. These peaks have disappeared, you see these helix which was present, these peaks have disappeared here which means they have gone into the assembly, into the associated state. Now this one is little bit of that is remaining. So so many peaks which are present here, they have vanished here because these have gone into the associated state. You cannot monitor those peaks because these peaks have vanished. Now if further go down to further another value of the DMSO, you see these many peaks have disappeared. All of these have gone into the associated state and this big helix is still remaining. And these ones are still, the N terminal anyway is a free thing to do, free thing. So this will remain till the end. So when you go further down, these are for standard typical points are shown here. See even from here, from these helix also, so many peaks have disappeared here. So small number of segments are seen in this area, some peaks are there but many peaks have disappeared. And this is the result of the association process. Now what happens with respect to the line widths and the relaxation properties? The relaxation properties are indicated here. So 100 percent, 90 percent, 85 percent, 80, 70 and 50 percent there. So the 50 percent again, the N terminal peaks are all seen and same here. And now as you go down from here, so this is the area which has on the basis of the relaxation properties is the R2 values, R2 indicates as I say the transverse relaxation rate. And then there is exchange going on because with the associated, dissociated exchange going on and that exchange produces the line grounding and the peaks will disappear and that is what is happening. So therefore, this step wise it indicates which peaks are disappearing, which peaks are in the exchange process and therefore that indicates the place where the association is going on. So as you come down, see the C terminal, this particular portion is the one which is very vulnerable and these ones are interacting and then of course you see these peaks have disappeared here. You come down to 85 percent, so many peaks have disappeared because these ones have participated in the association process. Going down further, so all these have gone, even this has gone, the C terminal has gone, some residues from here also have gone. So these have also gone into the association process. And then by the time you reach 70 percent, you already have vanished all these peaks which are here from here to here. So almost about 25 residues from residue number 25 till the end all the peaks have disappeared and 50 percent it is the same. Now this on the smile right side, it indicates where the helical propensaries are there and how the line width changes are happening. See this one is the N terminal which is present here. This has the flexible portion. Flexible portion this is always seen, this is always seen here in the unfolded state here and then of course it gets a little bit of a helical propensity when you come down in this N terminal also. And then some helical propensaries are seen for these residues which are in the interior of the box. This box is actually representing the associated state. So the residue numbers are given here, so which residues are going into the associated state and form helical propensaries which are remaining flexible without a structure. So as you go down further and further more helices are formed and more helices are getting into the interior of the aggregate and they are already disappearing. Notice when you looked at the CD spectrum of the entire aggregate it was mostly helical. Therefore eventually the protein goes into the helical state. So here all of this is helical everything is going into the interior of this aggregate and everything has disappeared. So this is how the process of self-position happens and this was extremely difficult to do it and this was possible only because of the pulse sequences which I described to you earlier. These were the HNN, HNC and pulse sequences which are able to study disorder proteins, flexible proteins because that one was able to identify the individual residues in all of these individual steps. Now let us look at what is happening from the structural point of view. So the same thing is indicated here. Now you look at this, this is the N terminal which has certain helical propensity here. So you go further to the next step. These helices start aggregating the transition, transiently they are aggregating. Then of course the proper structure is formed, the stable structure is formed, the helix is formed and these helices start aggregating here. This can form the helical aggregate in this process. Now in this process one can identify, analyze it a little bit more what sort of an aggregate is formed or is it in this way or they are oriented in opposite directions. Here we have shown that at this point we are not able to say that this N terminal and the C terminal they are all going parallel. Is that the way it is aggregating or it is any other way? So now let us look at that. If you look at this amino acid residues which are present in these regions. Now you plot here the electrostatic charges on this individual residues. The N terminal is has a particular charge and the C terminal has a red charge here. These are positive negative charges as we can see and if you lay this like this is a complementarity comes in here. It is because of this, this association is happening. The electrostatic interactions here, positive negative charges are coming close and that is what is causing the aggregation. See this aggregation which means these two chains are not going in the same direction but they are going in the opposite direction and that is what is shown here. See the aspartate is a negatively charged residues, glutamate is a negatively charged residues. What is present here? N terminal this is the lysine which is a positively charged residues. The positively charged residues is coming close to the negatively charged residues and all of this therefore this is the complementarity of the charges which are there. Same thing is happening here. So N terminal lysine 6, 8 you put them in opposite orientations. This portion is this and this portion is this. Put them in opposite orientations. This leads to the mechanism of the association process and that is what is shown here. So you draw adjacent chains in opposite directions. N terminal to the C terminal, N terminal to the C terminal and N terminal to the C terminal seems like that. Notice the extension presented at the C terminal is quite small compared to the extension presented at the N terminal. So therefore you see this here and then you further go down and then see further helix is formed. This is one particular helix. Now you form another helix and to go next step. So once again you see these helixes once again you will have to put them in opposite directions N terminal to the C terminal entire C terminal is covered here. Entire C terminal has formed helical structure. This is a stepwise process as I shown you earlier and this also is helix structure and a large N terminal segment is flexible. So you draw that here. So the N terminal is small helix, a long helix in the C terminal once again from here put in opposite direction N terminal short helix and the long helix and these are complementary to each other and therefore they will form an association associated state. Now you draw that picture in this manner to show the association which is happening. You lay them one over the other. See there is nothing almost at the C terminal. C terminal is completely in the helical form. The N terminal is hanging. So when you do this you get a cylinder you lay them one above the other you get a sort of a cylinder which means it is like a rope. See now the GED is now forming a rope and you have this flexible N terminal on either side. So these are like the arms. These are like the arms of this assembly of the helical associate. How is it useful? This is useful because this GED oligomer which forms a rope which binds to the membrane at the budding vesicle. Budding vesicle at the neck of this is all lipid membranes. Therefore and the lipid membranes they have the negative charges and we have here residues which are positive charges and they will bind to the phosphate groups of the negative charges of the lipid membranes. But this will associate in the as a form of a neck and wrap around the neck of the vesicle. So that is how this entire rope is able to bind at the neck in a in the type manner. Now from this of course we can incorporate this in taking the entire entire dynamite and then you will have the organization of different domains in the dynamite assembly. And the red ones are the GEDs and this is the assembly and other domains you can put them aside and then you can form a rope with this kind of a thing various domains which are putting. And this comes from the certain other biochemical evidences as well which domains are interacting this comes also from certain other biochemical evidences put that together here and say you generate a model of the organization of dynamite in this form of a rope. And that is what is shown here. So this is the neck of the envisioning vesicle this is the membrane, this is the membrane surface here. And you draw this like this the previous model which I showed you here you have the GED domain middle domain pH domain GED and and this here this is the GED in all of this this is the alpha associating into this D is the GED, A is GED domain and C is the pH domain these are from the two different molecules to opposite sides. Therefore this will wrap around this vesicle here this is the dynamite tube therefore we say this is the kind of a dynamite tube which wraps around the vesicle of the neck of the envisioning vesicle that is how the process of endocytosis comes. Of course for the biological process to happen for the separation of this thing the GDP energy is required GDP hydrolysis works there to generate the separate out the budding vesicle. So, that is how we study association process. Now we will show you one example how the dynamics and the structure are important for the biological function. Already showed you in the case of HIV Protease I will show you one more example here and this is with regard to this particular protein called as dinin light chain protein. So this is the trafficking protein it carries the cargo from one side of the one part of the cell to the side of the cell it walks on the microtubule here this is the cargo molecule and this is the small dinin light chain the particular portion of this protein here is actually binding to the cargo and carries it along on the microtubule and the structure of this one is shown little bit more detail here. So there is this small molecule here DLC8 this is approximately about the 1890 residues here the structure of this protein is shown here this is this fellow which actually binds to the cargo in the dimer form this actually remains as a dimer at pH 7 as pH 3 it is monomer because pH 3 it is not functional because that biological pH it is a dimer and it is the dimer which is responsible for binding to the cargo here and that is how it carries it forward and the monomer is not able to bind what does that mean if we change the pH of course you cause a transition in the structure of the molecule and it can affect your efficacy of binding the cargo and this is the biological important I will show you this illustrate this to you how NMR was helpful in understanding this this was actually studied using a particular cargo here with a small peptide here somebody had this peptide here as a cargo and you have a dimer here. So this is a what does the protein consist of it consists of helices here there is a helix helix and then of course you have a beta structure there okay so largely it is helical protein and you have the loops and the beta sheets going on so at this point there are the beta structures and this is the dimer interface this is beta 3 beta 4 loop at the dimer interface the cargo is bound see held together the cargo is held together at the dimer interface by this kind of interactions there okay therefore any perturbation that happens at the dimer interface will affect the binding efficacy any perturbation that happens in the dynamics of the dimer it will also affect the binding efficacy okay. So let us see what happens now we look at the dynamics of this protein this can be studied by relaxation data by the T1, T2, NOE and all of those ones which will be covered also separately in Professor Roshan's lectures and so here you see the red ones are the areas where there is a dynamism okay this is that one particular pH this is that another pH this is that pH 7 this is that pH 6 so at slightly lower pH 6 or 6.5 so there is there is a change in the dynamics in the protein as we change the pH of the solution okay and this has important implications this is what we will see. So pH 7 this is the DLC a dimer and this is a very beautiful spectrum as you can see all the peaks are very well resolved okay and you want to focus our attention on this particular all these few peaks I am showing only the few peaks but things can happen at every other place as well just to illustrate you know this is the particular four peaks you look at it this is G and this is serine and this is a spudate and this is isoleucine so this at pH 7 this is a very well resolved spectrum. Now if you add the peptide which is the cargo add the peptide to the cargo what happens there is a kind of a peak movement because the peptide is binding when the peptide is binding of course there is a structural change there the chemical shift changes are happening so it is coming this peak has moved here this peak has moved here okay and similarly this peak has moved here this peak has moved here I am only showing you the four peaks there are changes in other places as well but just to illustrate this peaks four peaks are picked out. Now you do the same experiment at pH 6 you change the pH of the solution you bring down to the pH okay at pH 6 what happens see some of these intensities come back here okay some intensity has come back here and some intensity has come back here too what is the meaning it means that the binding efficacy of the peptide has reduced so certain amount of free protein is produced earlier everything was bound the peptide was bound and once you change the pH the binding efficacy has reduced therefore this peak has come back here okay so this has an important implication for the biological function as to how this protein can carry the cargo so this is the pH switch for cargo trafficking if we change the pH slightly then the binding efficacy will change so why is it important because cargo trafficking meaning what so the protein has to bind the cargo at some place and release it another place okay what could be the mechanism for that and we are saying here that if it has to be released is a small change in the pH can do this but how can the pH change happen pH can happen due to some signaling the signals can come from outside to the system as a result of which there can be change in the dynamics and this is the areas where there is dynamics changes this happens because with the change in the dynamics when there is a change in the dynamics of course the binding efficacy will change why does the binding efficacy change when you change the pH because of the protein becomes more dynamic so when it is so then it is not able to bind it in the same way as it was doing when it was a pH 7 okay so therefore here the dynamics and the structure are important for bringing out a biological function therefore we say this is the pH switch for cargo trafficking there can be other signals also various other signals are also possible that okay some other interactor will come and then caught release and things like that but even without that even the small change with respect to the pH there can be acidity and things like that various sort of things can happen and that can cause a change in the condition from one part of the cell to other part of the cell and then you will have the trafficking possible okay so I think I have come to a close here so with all of this so we complete this particular portion of applications of NMR to different aspects of biological function we can describe the various aspects of structure determination we described the protein folding pathways and we described the methods earlier and from the protein folding pathways we looked at the association with the large assemblies and the large assemblies how to investigate using different techniques and the different techniques are of course to be used carefully at a particular magnetic fields not to lose the intensities and then the association process can be investigated by using the special sequences which we described these were the three dimensional HNN HNC and based experiments there are quite a variations of those which will which are quite useful in addressing different kinds of protein systems okay and then we also looked at how one can use this to study the folding pathways we took two examples one we took out the sumo protein then we looked at the HIV protease and demonstrated that cooperativity is an important phenomena and which can be understood using NMR how cooperativity happens this is the process which happens in the protein folding process and this can be analyzed and understood which portions are cooperating in bringing about the final native state and then we looked at the how biological structure protein structure and dynamics is responsible for the biological function I think with that we will stop and we sort of end the course here.