 So, just a brief recap of the previous class and we talked about the various amino acids, 20 different amino acids and what are the chemical shift ranges of the different protons in the different amino acids. These are indicated, these plots here for the different amino acids you have different ranges of chemical shifts for the different types of protons. So, we also talked about the different proton types, the nomenclature as was discussed in the previous class. And these ones show up in different kinds of two dimensional spectra. A typical one is shown here, this is the chemical shift in the folded proteins of the Toxy spectrum. So, here only one particular region is shown here. So, the amide protons to the various alpha protons and the beta protons and things like that and also the side chains here. So, and a whole COSY spectrum is shown like this here. So, it shows this is the area of the amide protons and the aromatic protons and you have the correlations in the COSY spectrum to the alpha protons here and then you also have from the aromatic protons to its own beta protons and so on. Then of course, when you go here then you will have the correlation from the alpha protons to the beta protons and then from the beta to the gamma, gamma to the delta and things like that. So, that is how the connectivities of the various protons are established in the COSY spectrum. The same thing will be available in the double quantum filtered COSY and the Toxy goes a little bit further and it establishes a relay of information in the spin system. So, here a comparison of the COSY spectrum here, a particular segment which is showing you the phi peaks here and these phi peaks actually are also present here in the Toxy spectrum, but it shows a relay to other protons and that is indicated by the different peaks which are present at the same chemical shift here. For example, for this particular peak here, there are others which are present here which shows the correlation to the other protons which are connected to this proton through a network of coupled spins. Similarly, for this one you also have a correlation here. So, like that for every proton there is a relay that happens in the Toxy spectrum that establishes the spin system network. And this is the another region of a particular spectrum, you have the COSY here and this is the Toxy spectrum here and you can see a full network of correlations in the Toxy spectrum only near neighbor interaction the 3 bound couples or the 2 bound coupled ones are shown in the, are seen in the COSY spectrum. In the Toxy spectrum, you have a whole range of connectivities from the alpha to the beta and then alpha to the gamma, alpha to the delta and so on so forth that will appear because of the relay that happens in the Toxy spectrum. This is what we have seen earlier when we discussed about the methods. So now we come to the next step that is the information in the J correlated spectra. So what do we use ultimately our objective is to obtain the so-called frequent specific proton resonance assignment in proteins. We are allowed to be able to assign the individual protons to individual residues. There are different types of residues which we discussed there. So we have to establish the networks of J couplings in the individual residues. This comes from the COSY double quantum filtered COSY or the Toxy spectra. This is known as the spin system identification. What is the meaning of the spin system? So let me also just briefly give a recap of that one. So if you have a 3 spin system, you may have a system like this, you may solve this is the AMX or if you have a linear system AMXQ and so on so forth, these are called as the spin systems. There are couplings at each stage, there is a coupling here, coupling here and a coupling there and here there will be coupling there, there is a coupling there and there is a coupling there. So all of them are in that way connected. So therefore this is known as the spin system. What type of a spin system is present in your spectra? So if you have a protein chain which consists of various amino acid residues, for example if I want to write an amino acid chain as A, T, G, C, L, Q and so on so forth, each one of them has a particular type of spin system connectivities and that is called as the spin system identification and this is known from the COSY and double quantum filtered COSY. Actually many of these amino acid residues may belong to the similar type of spin system. That is why we need to classify these amino acid residues as AMX system or a long side chain. So you have the classification of the residue types, AMX type that means it will be something like this here or the long side chain it may be something like this here or the glycine which it does not go too far or there are some residues which contain the methyl, these are typically the leucine, isoleucine, alanine and threonine valine these ones are the methyl bearing residues they have produced characteristic patterns in the COSY and the TOXY spectra. So therefore from the COSY and the TOXY spectra what we do is that a particular chemical shift what type of a spin system is present whether it is an AMX system or is an AMX Q type of a system or AMX QR kind of a spin system how many protons are connected to in the network. So that is what we establish in this the long side chain means there are many protons which are connected in the network glycine has only two protons and the methyl bearing residues also may have a long side chains all of these can come inside this network of a spin systems and we classify the amino acid residues in that. Then after we have done that we have put the polypeptide chain and we do not read it IK polypeptide chain in this we will read it as a spin system sequence that is the three spin system long side chain sequence glycine and then of course again this long side chain again a small spin system and so on and so forth. So you will categorize them on the basis of the spin systems therefore you set the writing the protein sequence in terms of the classes there will be several classes as we indicated here these are the types of the amino acid residues. Eventually we do not know which residue is where so you may have several glycines in your polypeptide chain several phenylalanines there will several tyrosines all of them belong to a particular type of a residue of the spin system therefore all of them belong to the same whether you have glycine whether you have a tyrosine or phenylalanine they both belong to the same type of spin system or you have the threonine is a different spin system alanine is a different spin system and likewise the long side chain once suppose you have the arginine or the lysines or the glutamine the glutamine these ones have long side chains so they will classify them as long side chains that is what you will write they will write the protein sequence in terms of the classes. That is the first step this information is obtainable from the COSY DQ of COSY and TOXY spectra because these display the J correlations the J coupling correlations notice here of course that we only have these things from the within the same amino acid residues these ones do not connect one residue to the next residue on either side therefore this is within the same amino acid residue what sort of spin systems are present and what are the correlations established. So from the COSY spectrum you identify that okay now if I go back here so I will say for looking at this this may be an AMX spin system this is an AMGlycine spin system or this is a glycine spin system or this may be a again an AMX spin system and like that. So I will say on the basis at the particular chemical shift I will put this as a chemical as an AMX spin system or a long side chain spin system or the methyl bearing residue spin system etc. So this is how you obtain this information from the TOXY spectra. So this is the COSY and to compare that with the TOXY here you get a stuff you get the connect the connectivities and on the basis of that you say okay now here it is a system which has a long side chain there are many cross peaks originating from this particular diagonal peak here you have a peak here a peak there and a peak there and a peak there. So therefore many correlations are present that means that particular spin system is a long side chain ones. So it has the alphas the betas the gammas and the deltas and so on so forth. So you first classify your polypeptide chain along these lines okay here it is a long side chain the long side chain short side chain methyl bearing AMX spin system and so forth. So that is what is called as writing the protein sequence in terms of the classes. Next we have to connect these spin systems sequentially. So we have these neighboring residues near river connections the near neighbor connections these do not come from the COSY spectra or the TOXY spectra there are no J couplings there and there are also no relays okay. So therefore this information will not be obtainable from the J correlated spectra. So we have to resort here to the nosy spectra the nosy spectrum displays distance correlations and there are short distances between the near neighbors and there can be short range connections over a longer distance in the sense that okay three residues four residues apart or there can also be longer range connections which are because of the folding of the protein there may be things which are coming which are 10 residues 15 residues 20 residues apart and you may have that sort of the connections also because the protons come close by in space and that is what the nosy spectrum displays. The nosy spectrum displays those proton-proton correlations which have short distances less than 5 angstroms. So therefore we have to distinguish all the three different types of connections here first thing is to use the near neighbor connections to identify the sequential connectivities. The individually you walk from one polypeptide chain one residue to the next residue along the polypeptide chain. So these are the two steps which are there and then once you do that you obtain the sequence specific resonance assignments in proteins okay. Now what sort of how do this spectra look like for the different amino acid residues this obviously depends upon the chemical shift ranges. Now here again I have put in the various amino acid structures there and the corresponding type of correlation you will see in the COSY or the TOXY spectra for example for the histidine ring. So where is the histidine here? So we have the histidine aromatic residues the let us look at where is histidine here, arginine okay the histidine is here okay. So now you see this has two protons there is a proton here and there is a proton there. There can be correlation between the amide proton to this proton and there can be correlation sometimes you will have correlation from this proton to this proton correlations you will see that sort of a system. So you will have the 2H and the 4H which are the 2H 4H this is the 2H this is the 4H. If you are look recording the spectra in D2O so when you are recording the spectra in D2O the amide protons do not show up okay or the amino protons or the NH protons do not show up because they all exchange with water exchange with the D2O and they will not be present. So these spectra are recorded in D2O so when we do that then of course you will see the correlation from this proton both these protons are attached to the carbon C2H and C4H those are these two positions okay and you will see correlation between them. So this will become like an AX suspense system. Similarly the tyrosine ring let us look at the tyrosine the tyrosine is the among the aromatic residues right the aromatic residues phenylalanine and we have this phenylalanine and threonine let us look at where is the tyrosine okay now here is tyrosine okay this is Y. Now how many protons are there? So this OH of course is not visible there are 4 protons there typically these two may be these two will be equivalent and these two may be equivalent but they may not be also. So if they are not equivalent then you write it as AA prime XX prime but if they are equivalent so what are those positions? This position and this position if they are equivalent then they will say this is in A2 and this and this if they are equivalent then it is a X2 so it can be A2 X2 spin system or if they are non-equivalent then they may be AA prime XX prime spin system and the non-equivalence can arise in the protein structure where there is a free rotation is restricted and therefore they may see different environments and their chemical shifts can be different. So therefore if we have what is drawn here is when the two are equivalent their chemical shifts are the same but their coupling constant may be different so though you may write in general as AA prime XX prime to be more general so but the chemical shifts of these two are the same therefore they are typically represented as one chemical shift here and another chemical shift there because we have used the same symbol A okay so the chemical shifts are the same but the coupling constants may not be the same that is why you say make them as magnetically non-equivalent but chemically equivalent when you have that of course there is only one coupling so the coupling is from this proton to this proton and that is this you will have from the in the aromatic ring you will only have this but of course in the in the backbone you will have the beta protons also the beta protons will show the alpha to the alpha to the beta 1 and beta 2 what is shown here is only the ring the ring protons what how are they connected in the ring okay these are special these are aromatic protons these are special they have the ring and therefore what is shown here is a connectivity in the ring now if you go to the phenylalanine there is phenylalanine is here now this says 5 protons there once again there can be equivalence here equivalence there but the third one will not be equivalent so therefore you have here 2, 6, 3, 5 and 4 so the 4 is the central fellow here and the other ones are 2, 6 and 3, 5 and you will see correlations you will see from 3, 5 to 2, 6 so here this is 3, 5 to 2, 6 you will see this cross peak okay 3, 5 to 2, 6 this cross peak is the prominent one and this 3, 5 those ones are here these are the 3, 5 and those ones show to the 4 therefore you see that cross peak also here you do not see 2, 6 to 4 2, 6 are these are these and you may not see the cross peak here to the 4 so therefore you do not see the 2, 6 to 4 but you will see from 3, 5 to 4 and 3, 5 to 6 you have what is shown here is on only one side of the diagonal but these peaks will also be present on the other side see these 2, 6 to 4 it can be a very weak one this may not be present but it may be seen in the toxic spectra these may not be seen in the cosy spectra this peak but they may be seen in the toxic spectra therefore this spin system becomes A A prime, X X prime and M and that is the one of this single fellow which is the 5 and that will be this the 4 this will be this M and these 2 are the A A prime, X X prime these are the 3, 5 and 2, 6 and this one is your M and similarly if you go to the tryptophen ring the tryptophen ring is this one here and you have the correlations of these protons there big empty circles are the ones which are seen in the cosy so you will see from 5 to 6 and then you will see from 5 to 4 then you will see from 1 to 6 and that is the nomenclature which if you remember that is represented them as the various alpha, beta, gamma etc but within the ring the nomenclature goes by the numbers in this one so you will see this sort of a pattern for the tryptophen you will see these 3 peaks in the cosy spectrum but in the toxic spectrum you may see this intermediate peaks as well you may see this peak, you may see this peak and those ones will be present in the case of tryptophen this 2 proton this one actually stands alone and this does not have any coupling to anybody so that is a single peak which is which will not be seen that is you will have this one here this proton there on the side and that does not have any coupling to any other proton therefore you will not see any correlation from this proton to any other proton and all others are in the 6 membered ring and you will see correlations between the protons in the 6 membered ring. Now we go further to the other side chains so you have here valines the side chains are indicated here already indicated here so you have this say gamma, gamma 1, gamma 2 so this is the valine valine has this there is a methyl here so you will see this sort of a pattern beta to the 2 gamma so it is coming from the alpha the alpha proton is here on the side alpha is not shown only the side chain beyond the alpha proton is shown you have one beta proton to that are attached to 2 methyl gamma 1 and gamma 2 and these are the 2 these are the 2 methyl this is NH T alpha CO OH and this is the C alpha this is the C beta and that C beta there is one proton there and it is connected to the 2 methyl there so that is shown here so you have the beta proton there and these are the gamma 1 and gamma 2 and this is the alpha proton. Now from the alpha proton you are seeing to the beta proton that is this the alpha proton is here on to the C alpha which is present there to the beta proton you see this peak here and from the beta proton you see to the 2 methyl from this beta to these 2 this and this correlation you will see from beta proton to the 2 methyl here and you do not see between the 2 gamma methyl because there is no coupling there therefore you will not see that cross peak loose in if you see again it has the alpha to the beta and then beta there are 2 beta protons you see to the both the beta protons alpha to the beta 1 and beta 2 these are the 2 betas and then from the beta you see to the gamma there is one gamma proton there so on the gamma you see so both the beta proton show to the gamma and from the gamma you see to the 2 methyl there are 2 methyl on the one is called as a delta 2 delta methyl some gamma proton you see to the 2 methyl you see to this peaks there and of course if you are looking at the toxic the dashed lines and dotted lines indicate that these you may see the additional peaks when you go to the toxic spectra in the cosy you will see only this or in the double quantum filtered cosy you will see only this empty circles which are indicated there so you will see this cross peaks and if you go to the toxic you might see this individual correlations as well. So similarly isole you see has this network of couplings there you have the from the alpha proton to the beta proton from the beta 2 you see to the gamma 1 and gamma 2 there are 2 gammas there beta to the gamma 1 and gamma 2 and these gamma protons are connected to the methyl to the delta and therefore you will see from the both the gamma protons you will see to the methyl. So that is how this spin system goes for these amino acid residues and likewise for the serins and other these are called the ABX spin systems or the AMX spin systems because this has the side chain has only alpha to the beta and to the that is all alpha beta there is nothing beyond the beta proton for example you can see here so serin has alpha to the beta and there is nothing and these ones will not be seen the aspartate similarly to the beta and you do not have any other coupling there these ones are gone asparagine this alpha to the 2 beta protons alpha proton is there on the side when the C alpha is there and so similarly the cysteine alpha to the 2 betas here the tryptophan this alpha beta proton region is also shown the aromatic ones also have this alpha to the beta we talked earlier about the rings here we only talk about within the ring what is present there now you see including the alpha you will see alpha to the betas the 2 beta protons the 2 beta protons and so on and there is no coupling from the beta protons to the aromatic protons therefore you will not see cross peak from the aromatic protons from the beta protons to the aromatic protons these appear separately and they can they have to be identified differently now all these are there are these many 8 amino acid residues which we classify as AMX spin systems or sometimes ABX spin systems so because there are 3 spin systems so wherever this serine cysteine asparagine is present in your polypeptide chain you may call it as a particular type of spin system that is called as the J spin system J we represented as J J J J J J wherever this is present you write it as J okay and the long side chain ones are represented differently so you have this sort of spin system and what you have here only alpha to the beta beta 1 and beta 2 and then you also have a cross peak beta in the beta 1 and the beta 2 protons so this is only one side of this diagonal is shown and you have this cross peak there is here the Toxy and the Cozy do not make a difference at all because there is there is a beta 1 and beta 2 and there is no the relay so there is a direct coupling here and all of these peaks can be seen in the Cozy, DQ of Cozy and Toxy they will be identical okay now this is another type of spin system as indicated these are the long side chains from the alpha you go to the beta beta to the gamma gamma to the delta and then delta to the further ones epsilon and things like that okay so there are long side chain ones so how many are there lysine, arginine, methionine, glutamine, glutamic acid, proline these are called as the long side chain ones because the network goes quite long you go from the alpha you see to the 2 betas 2 beta to the 2 gammas 2 gammas to this ones there of course you may not go beyond here something these are the amino protons and these will exchange out and you will not see so it will be seen only up to this point if you see lysine from the alpha to the 2 betas 2 betas to the 2 gammas then to the 2 deltas then from the you go to the 2 epsilon and after that you do not see because this one goes to the group and this will be exchanged out in the d2o spectra arginine it is similar alpha to the 2 betas beta to the 2 gammas gammas to the 2 deltas and after that you do not see okay so and therefore the lysine has one more non exchangeable proton compared to the arginine whereas the arginine has more exchangeable protons these will be seen more in the h2o spectra there also you will see one of them that is typically here but these ones also typically exchange out in water because of the exposure and the methionine is alpha to the 2 betas to be to the 2 gammas and then of course you do not have any other thing there is a CH3 group there but it has no coupling to any of these one this is the long the 4 bond and you will not see a correlation between these here and the glutamine glutamine has once again alpha to the 2 betas beta to the 2 gammas and then after that you do not see anything there and because these are exchanging out with the water and the glutamate is also similar glutamine and glutamate are similar and now the proline the proline is interesting you see alpha to the beta okay there are 2 betas 2 betas to the gammas 2 gammas and the gammas to the deltas here and that is that is it what we have so therefore this is a closed ring here so and you will therefore because the ring closure of course its chemical shifts are also different and how does it look in the spectrum and the spectrum I am showing you here the alpha proton region alpha proton region is here this is typically alpha to the 2 betas now you see that the betas are of are more of field compared to the gammas in these residues glutamine glutamine methionine these the betas are upfield to the compared to the gammas why is it so because these gammas are attached to either a sulfur or oxygen or a nitrogen therefore they get downfield shifted in these 3 residues okay and therefore the betas are above and the gammas are below so you see from the beta to the 2 gammas alpha to the 2 betas and from the both the betas you see to the 2 gammas therefore you have 4 peaks there and you also have the beta beta cross peak okay and now in proline in arginine so you have the alpha to the 2 betas and now this one is in the middle the chemical shift ranges look at the chemical shift range that is important to notice here how the chemical shifts vary depending upon the structure of the side chains there so the 2 betas are here and the gamma is above to the betas are connected to the gamma by these 2 cross peaks and the gamma is connected to the delta which is down here which closer to the alpha proton here gamma is down here so this characteristic patterns are very interesting the same thing happens with the arginine and in the case of why the delta is below because why the delta is below because they are attached to the nitrogen see both in the arginine as well as in proline the delta proton is attached to the nitrogen and therefore this comes much downfield and is closer to the alpha proton therefore this pattern is very distinct from the other one there although they are all long side chains but the patterns of the peaks are different there and lysine if you see the alpha to alpha to the betas which are down here and beta to the 2 gammas the gamma the gammas are here beta to the 2 gammas these peaks there of course you have the gamma to the delta which is very close to this one often you may not be able to distinguish between these because these are too close and then from the delta to the epsilon now the epsilon is distinct epsilon comes very far away because it is attached to the nitrogen so this comes quite downfield there so this is how you get a very distinct pattern for this spin system so these are called as u spin systems and because these ones are long side chain ones they are called as u spin so this is the nomenclature which was adopted in the in the book by professor Wittrick and these pictures are actually taken from the Wittrick's book all of these are from the Wittrick's book which is NMR of proteins and nucleic acids and that is published quite a long time ago and he was a pioneer in doing all of these assignments and the protein structure determination for which he of course won the Nobel prize in 2002. Or the next step next step is to do connections from one residue to other residue and this will come from the nosy spectrum. I think we will stop here when we take it to the next class.