 So, we have been discussing about multidimensional NMR methods, we discussed the different types of 2D correlation experiments and last time we also talked about the heteronuclear experiments and I want to show you some of those spectra and illustrate how these heteronuclear experiments are extremely useful, how we take it forward to three dimension, four dimensional experiments as we also mentioned earlier but we will elaborate on that one as we go along, different kinds of experiments are can be done. Now, one of those which was most crucial experiment in this endeavor was the so called HSQC, heteronuclear single quantum correlation experiment and that is HSQC and to this one what I am showing here is the so called proton N15 HSQC of a protein which is labeled as FKBP, it is a small protein of about 1090 residues and you can see here peaks coming from the individual amino acids as they are labeled here. You can see the various amino acids are labeled with their one-letter code, so for example there is a glycine here, there is glycine here, all these are glycine then you have serine here, glycine, G, glycine, valine and incidentally these peaks which are coming here these are coming from the side chains, side chains of glutamines and asparagines, side chains also have the NH2 groups, CONH2 groups of the glutamines and asparagines have the CONH2 group in the side chain and they also produce peaks here and they appear as pairs here, so whereas all the other peaks which are appearing here these are from the backbone of the protein and you can get this one peak per residue, I mean every amide proton has a connection to its own N15, therefore you can see here such a good distribution of the peaks, this is one of the very well behaved, very well folded proteins. Now if you can imagine here how this experiment has been extremely useful, see suppose you were to take a projection here, take a cross-section at a particular NH position in the proton spectrum how would it look like, suppose I were to take the amide proton at this chemical shift and you see how many peaks are here, 1, 2, 3, 4, 5, 6, so many amide protons are present at this particular position, this is what I illustrated to you last time that when so many amides are present they will all come at the same line in your correlation spectrum of the toxic or the nosy and things like that but now you see all of these amino acids have different N15 chemical shifts and therefore they get separated here on this particular N15 axis here. Similarly, if you took here there are so many amide protons at the same NH chemical shift and they are fortunately they have different N15 chemical shifts, of course similarly if you were to take the particular N15 chemical shifts some way for example here, so then you will see so many N15s at the same point, so you have here suppose I took this, you have this one, this one, this one, this one, this one, this one, this, so many N15s at the same point N15 chemical shift but then they have different amide proton chemical shifts therefore they get separated out quite distinctly in this two-dimensional correlation spectrum. Amide proton N15 correlation spectrum that is why it is called as heteronuclear single correlation spectrum. Therefore since you are able to count here the number of peaks distinctly one for each residue this the number of peaks will be almost equal to the number of residues in your protein, barring prolinx. The prolinx do not have the amide protons therefore you will not show those cross peaks and therefore this experiment is called as the fingerprint, fingerprint of the protein with regard to its primary structure that is the amino acid sequence fingerprint of the protein with regard to the primary structure. You can just count the number of peaks here first of all you count the number of peaks to find out whether you have got your protein properly or not or there is something else is happening. If there is aggregation going on in your protein then you will see that some number of peaks will not be the same some peaks do not appear then this will also give an indication as to what is happening in your protein whether the protein is aggregating and if it is aggregating there will be only few peaks which will be seen and these will correspond to the flexible portions of the aggregate. Therefore this also you make use of to find out the status of the protein in your solution and then of course you have to devise conditions how to break these aggregates and how to get single thing and this is what extremely is a useful technique and we will show you how this has been used to identify or determine the folding pathways in proteins or aggregation pathways in proteins. This also has been extremely very well character useful procedure for this. Now of course what you need for this you need a labeled protein. So you need here a N15 labeled protein you cannot do this in at natural abundance and in fact this is how this was these experiments were not done earlier when the methods were not available for labeling the proteins N15 labeled proteins are required for this and this is now possible because of the recombinant technology. You can produce this protein inside equalized bacteria and you can put your gene of interest this kind of a construction construct and the protein will be expressed in the cell you feed the bacteria with ammonium chloride which is N15 labeled and then it will and no other source of the nitrogen in that one. Then it will incorporate the N15 in every amino acid it synthesizes and then in the every protein it synthesizes therefore you will naturally get N15 labeled protein from the bacteria you will use a biological machinery to label this protein with N15 and then of course you will have to devise methods of purifying your recombinant protein and then record the NMR spectrum. So now having done that so as we also discussed earlier that we can make use of this strategy and combine this with the other 2D experiments like the Toxy and the Nosy. The HQC spectra gave you only the amide proton chemical shifts it identified the amide proton chemical shifts and the N15 chemical shifts but the amino acids have other protons as well along the side chains and along the backbone the alpha protons to the beta protons to the gamma protons and things like that. So therefore you should be able to identify those ones and also if you are going into the structural part then you must be able to identify which protons are close by and this is what is done by these particular kinds of experiments where you have this HQC part from here HQC which we have discussed earlier this is the HQC part you combine this with the Toxy spectrum here this is the Toxy mixing sequence until here it is the HQC up till here then you make combine with the Toxy part here and then you record the spectrum in T2. Therefore what will appear along the T1 axis the T1 axis the information that is present is the N15 or the C13 here in the case C13 as a pulse sequence is written but the same thing is true for the N15 as well. So therefore you can this will be the carbon 13 chemical shifts or this N15 chemical shifts in the case of proteins but in other molecules you can do carbon 13 chemical shifts here I will show some examples of this one then after this Toxy you can record this spectrum what happens during the Toxy period the magnetization which has come back on a particular starting proton will get relayed to other protons through the Toxy mixing. So this Toxy mixing relays as we discussed before to other protons through the sequence therefore at a particular carbon 13 you will see all this protons which are connected in the network. Similarly in this particular example until here you have the HQC spectrum so the C13 proton correlation here and then after this portion which is added here this is the nosy so you have the mixing time here the tau m is the nosy mixing time. So during this period you have the transfer of information happening between the protons which are close by in space or if there is a chemical exchange happening that also can be happening here. So whichever process is happening you will see correlations for those particular protons therefore this has a structural input. So while this is useful for identifying the spin systems the assignments this has a structural input. It also has input from the point of view of assignments because the near neighbor interactions will provide you the assignments and it will also provide you the structural information as in your depending upon the molecule what you have. Let us see here is an example here. So here is an example of a small molecule this is for trichinine this is some kind of a drug I guess. So this is a molecule which has been investigated in detail see the very complex molecule you have so many protons here all the protons are numbered here see you have 21, 22 so many protons are there is a carbons these are all carbons so many carbons are there and they starting from 1 here 1, 2, 3, 4, 5, 6 so many carbons are present in this molecule and there are correspondingly so many different protons. Now to determine the structure of this to assign this individual spectrum it is extremely complex so we look at this chemical shift this is the one dimensional spectrum of this molecule so you have so many peaks here of course this is the same here as well and this side is the HSQC toxy spectrum this is the HSQC nozy spectrum there is a big difference between these two and you can see at the one which are the dark ones here focus on the dark ones here the red black peaks these are the HSQC peaks alone these are just the HSQC peaks these correspond to you can count here the number of peaks this will correspond to the number of carbons you have in your molecule. So each one of them here corresponds to one particular carbon each peak corresponds to one particular carbon and you have here the number of peaks which are equivalent to the number of peaks in carbons in your molecule of course if you have a quaternary carbon that will not show up here because wherever there is a proton attached to the carbon only that carbon will show up here so you can have as many carbons identified there. Now what you do at the same carbon positions you see this toxy so you have the whole range of protons which are attached okay so from here to here there are so many protons which are connected to that so different carbons there and from the carbons you have a relay to the protons which are attached therefore you can identify the protons which are connected to these carbons okay so you see here these are once which are connected in the coupling in the network okay you can look at these peaks here so which are the ones which are connected okay the numbers are indicated here so this assignment has come as the result of analysis of this spectra so you have the carbons and you have the connected protons to them and you go to the next carbon which is connected to that then you walk through the spectrum to identify the entire network of carbons which are connected to each other so that is how you get a sequential walk or a walk through the spectrum through the toxic spectrum to identify the various carbons in your molecule okay what about this this peak here you see you have the HSQC spectrum which are black and the nosy peaks which are present here these are appearing in red the red means they have a negative peaks these are negative peaks why are these negative nosy because this is a small molecule you remember in a small molecule which is less than few hundred molecular weights or few hundred molecular weights the tumbling is extremely rapid in those cases the NOE is positive if the NOE is positive in the two dimensional spectrum it will appear as a negative peak whereas the diagonal or not diagonal the self peaks appear positive and the NOE peaks will appear as negative therefore you can clearly distinguish which are the NOE peaks and which are the self peaks here that is why I said all these black ones are the self peaks of the individual carbon proton correlations it will tell you how many carbons are there in a particular carbon chemical shift and from those carbons you have a NOEs to the proton protons so what does that tell you that means these protons are having correlation through the distance dipolar distance and therefore you can identify using these you can figure out which protons are close by here because the NOE can happen between the protons and protons we are talking about proton proton NOE we are not talking about carbon proton NOE we are talking about proton proton NOE so let me let me go back to this experiment here you see the magnetization at this point is on proton magnetization is on proton so therefore we are talking about the proton proton NOE so therefore when I see here so the NOEs which are happening are between the protons if I have a carbon here it is attached to a proton and it shows NOE to another proton and it is attached to a different carbon then you will see that sort of a correlation here we can walk through the work through the carbon chain here from the proton-proton correlations appearing the particular carbon chemical this is just to illustrate how these experiments are useful for arriving at the assignments on the one hand and the structure of the molecule on the other hand ok. So now you can see you appreciate this better the how we can make use of three dimensional experiment now we talked about the two dimensional experiments we said at a particular I explained to you earlier at a particular amide proton chemical shift there are so many in that amino acids here so many therefore you are seeing so many chemical shifts so many peaks here ok this may be a toxic spectrum this may be a NOE spectrum whatever so you have so many peaks here at a particular n proton chemical shift now if you look at the proton N15 correlation spectrum you are seeing three N15 therefore there are at least three or certainly three because there are only three peaks there so you have three peaks which are present here and now you do what you do is instead of making a just a two dimensional spectrum hsqc spectrum hsqc toxic or something like that instead of spreading them along the proton axis extending the proton axis here like that we actually pull this spectrum along this axis here in the two dimensional spectrum what we did we would actually pull this in the hsqc toxic or hsqc NOE to include the proton chemical shifts more here but now we will restrict that to N15 the amide chemical shifts only but we will take the third axis to pull these peaks on to the different dimension the N15 dimension here so therefore all these peaks will now get distributed into the different planes in the three dimensional spectrum now if we need further resolution you can make use of the carbon 13 here then of course you will go into the 4D experiment and this will be quite a substantial thing to do now if you want to do carbon 13 then you will have to have a carbon 13 labeling as well this also can be done using the bacteria and use carbon 13 labeled glucose and you will get C13 and 15 label protein here is a typical example of a three dimensional spectrum here axis are not labeled with anything but I already showed you yesterday that in the previous class then you can have whatever nuclei you want here you can have you can have two proton axis or one carbon axis two proton axis or one nitrogen axis or one proton axis one nitrogen axis and one carbon axis so you can have all three different kinds of combinations there so now the peaks are distributed in a volume in a cuboid rather than on a plane therefore you take cross sections at various places you will be able to figure out what sort of a spectrum you will get what is the information present in your spectrum okay now we go to the next stage we go to the next stage what are called as triple resonance experiments so far we are talking about two dimensional experiments or three dimensional experiments where there were only two nuclei one was the proton other one was the nitrogen now you can have three different nuclei also right as I mentioned you can have proton and nitrogen and carbon what does this require this requires different kind of a treatment you need to have three different channels you need to have three different channels I will write this on the next slide here so in your these are the same slide given in the white background so that you some only only some experiments have been selected here so you need to have in your spectrometer you need to have a proton channel a carbon channel and a nitrogen channel so you need to be able to apply pulses along the three different channels okay so you apply various pulses there you apply various pulses and how it will and obviously finally you actually collect the data only on the proton channel so in between the magnetization flows through different ones which I will explain to you we take one by one and see how this works okay let us look at this particular experiment which is called as the HNCA what does this represent this experiment represents that along one axis you have the proton okay this is proton and along the second axis you have the carbon these are the C alpha carbons and on the third axis you have the nitrogen the red ones are the nuclei which you are going to see in a three-dimensional experiment that is why these are called as triple resonance experiments because you are making use of proton nitrogen and carbon three different channels okay and these experiments were described way back in about 1990s by different group authors here so I have listed those references here for quick reference seeing through that now this experiment it starts from the proton here and the magnetization is transferred you see how this is manipulated you manipulate the transfer pathway of the pulse sequence you go from here to here here to here or here to here and then come back here come back here and come back to the amide proton and detected this is exactly the strategy what we used in the HSQC toxio or HSQC nosy we went from the proton to the nitrogen and we came back to the proton to observe it now we are doing three different things here we go from the proton to the nitrogen and you also have the frequency label there and from the nitrogen you go to the carbon to the both the carbons here C alphas you also have a frequency label there so you need to generate another independent time variable and after that you come back here come back here and come back to this and you generate a detect the proton signal okay so now this is called as the HNCA now the next experiment here is HNCOCA there are so many different kinds of experiments here and the red labels the ones which are nuclear marked red are the ones where you have the frequencies label okay so in this case you have the proton the nitrogen and this C alpha notice we do not go to this C alpha we go to this C alpha how do we do it because we go we in this case we went directly from the nitrogen to the C alpha we transferred magnetization from that nitrogen to the C alpha here we do not do it we transfer from the nitrogen to this carbonyl we transfer to this carbon and from this carbon we transfer to this C alpha therefore there is no question of going to this C alpha at all this provides a kind of a directionality in our assignment procedure from this amide proton we go to this nitrogen from this nitrogen we go to this carbonyl from this carbonyl we go to this C alpha then we come back to this carbonyl come back to the nitrogen and then to the proton for detection we detect the signal on the on the proton okay so here therefore in the three axis what are the three axis we will have again proton nitrogen and C alpha but this is C alpha only now okay now we do another trick here this experiment you have the amide proton the nitrogen and to the carbonyl you label this carbonyl come back to the nitrogen and detect this on proton therefore we label these three here I have proton nitrogen carbonyl these are the peaks which will appear in your spectrum so likewise you have various possibilities of transfer pathways and this is manipulated by using different kinds of pulse sequences we are not going to go into that one but we see we know the principles how to transfer magnetization from one nucleus to another nuclei another nucleus and we simply apply those principles here so what we do in this experiment so we transfer from the amide proton to the nitrogen from this nitrogen we transfer to the C alpha here this C alpha as well as the S alpha this is the same as this this is the same as that now what we do from the C alpha from the C alpha we transfer partly to this nitrogen and partly to this carbonyl partly to this carbonyl and now we put the red label on this carbonyl we are not putting the red label on the C alpha as we did in this particular case so we use this is the mediator here as the C alpha come to the carbonyl and then we from the carbonyl we go back to the C alpha then back to the nitrogen same here C alpha carbonyl to the C alpha then to the nitrogen then here therefore these ones have different kind of information as you will see and similarly you can also do that on the side chains of the individual amino acids this is called as the HNCA CB or corresponding on the HNCO CB these are various experiments which are designed to walk through the amino acids different carbons in the same amino acid residue. So in this case we actually start from this beta protons here from the beta protons we start we come to this C alpha here and then from the C alpha we transfer to the nitrogen from the nitrogen we transfer to the amide proton and we detect the signals here. Therefore what are the signals which we detect here we detect C betas the nitrogens and the and the amide protons we finally detect this thing always notice we detect the signal on the amide we do not detect the signal on anywhere else because the amide protons are very distinct and we can easily identify those ones there in this particular case you again start from the C beta protons here from the beta protons you go to the C beta carbon from the path you go to the C alpha here and then from the C alpha you go to the carbonyl and unlike here from this C alpha we went to the nitrogen directly but here we do not do that we go from the C alpha to the carbonyl from the carbonyl we go to the nitrogen from the nitrogen we collect on to the amide proton and therefore this is called as and what is written inside here is what is written inside the bracket is these are the ones which are on the pathway these are there on the pathway but we actually do not detect those signals. Detecting means we have to have a frequency label and the T1 increment should correspond to that particular chemical shift and that is what determines which nucleus you are going to detect okay. So yeah and then the CBCACONH so these were the HN protons okay now here what we do we start from the C beta once again from here to here so we have from the C beta you come here from the C alpha also you start here you also start from the C beta so both the things will come here and from this you go to the nitrogen and from the nitrogen you go to the amide proton and you detect the signal here okay. So in these in these two experiments you can also start from the amide you actually in fact start from the amide proton amide proton to the nitrogen nitrogen to the carbonyl carbonyl to the C alpha from C alpha you go to the C beta and then from the C beta you go back to the C alpha then to the carbonyl and to the and to the amide proton and detect it here okay. So you can do that either way so therefore these kind of these are called as out and back sequences so different kinds of strategies one can use to achieve the your magnetization transfers okay all this the way you start and where you end will determine the way you design your pulse sequence okay from whether you want to start from the alpha proton or the beta proton or from the NH protons and where you would finally come back and detect it that is but ultimately you always want to detect on the amide protons in this particular experiment you see two amide protons here so you have this NH proton as well as this NH proton so you have this one's coming our guns again it goes through the same pathway go from here to here here to here and here and you also can go from here to here or you can go from C beta to this then from here you can go here then so these are all out and back pathway and from here you start from the C alpha you go to this nitrogen as well so you have transferred to the NH proton at this point okay. So this is explained that in a little bit more explicit manner in the few of these experiments here so you have HNCA, HNCO, CA, HNCO these ones provide a crucial assignment strategy here in this C VCA and CO NH experiment this is much more clearly indicated in this slide than in the previous one so in this experiment you see you start from the H betas from the H betas you go to this C beta from the C beta you go to the C alpha then from the C alpha you go to the carbonyl and in this you can also the some portion of the magnetization will come from the H alpha also because the H alpha and H betas which are a lot the difficult to distinguish with regard to the chemical shift the pulses will apply to both so therefore it will come from here to here as well and then from here to here as well both the things will happen then you go to the NH pro amide N50 and then to the NH proton and for detection here and in this particular case the CBCACO, CBCANH you have the starting from here H beta or H alpha you come down to the C alpha either a two step process here H H beta to C beta and then to the C alpha and from here all you go to the C alpha from C alpha you go to the nitrogen and and from this C alpha you also go to this nitrogen and you collect the signal as amide protons chemical shifts at this point therefore these will establish correlation what correlation do they establish they will establish correlations between within the same amino acid residue up to the C beta carbons but you see this experiment will also establish a sequential correlation because it has the two amide protons involved here of two consecutive residues this is one residue here and this is the next residue here so therefore you will establish correlations not only within the same amino acid residue but from one residue to another amino acid residue now these experiments are dependent on the transfer pathways and how is the transfer occur the transfer occurs through the J coupling and the J couplings are these are all one bond J couplings one bond J couplings and these numbers are indicated here see the one bond advantage is on one bond or two bond the advantage is that these are not dependent on the confirmation of your protein and these are always the same therefore there is no question of missing a correlation here in this kind of experiments here so you have this various coupling concepts that indicated here these are the carbon carbon coupling 35 hertz carbon proton coupling is 140 hertz this coupling carb this c alpha co coupling is 55 hertz and carbon nitrogen coupling this one bond is 15 hertz this two bond coupling nitrogen c alpha is 4 to 9 hertz and one bond nitrogen c alpha coupling is 7 to 11 hertz nitrogen to proton coupling is 90 to 95 hertz all of these do not vary depending upon the amino acid do not vary too much on the confirmation depending on the confirmation therefore you will not miss any correlation unlike in the case of noses where you can miss a correlation if the distance is longer if kind of a motion is such that you are not able to see a NOE but here you will not miss any peak therefore this is an extremely useful strategy to obtain sequence specific resonance assignments and for large proteins reasonably large proteins which you could not handle through the noses spectra because these are using making use of the amide N15 and carbon chemical shifts all the three chemical shifts are used and therefore the dispersion is enhanced and you will not miss any assignment so okay so we will stop here and we will explain a little bit more about the sum of these pulse sequences to give an idea of how the transfer happens in these pulse sequences as in illustration we are not going to discuss every experiment in this but I will take some two illustrative experiments to show you how the manipulation is done through the pulse sequence I think we will stop here