 So, let us continue with the description of the protein structures at more fundamental level. We said that the relative orientations of the amino acids depend upon some torsion angles described as phi and psi in the previous slide and we will examine more about that. Now, so here we have the peptide chain running like this schematically indicated here. So, you have the alpha carbon there is a carbonyl here. So, NH the chain is running like this, the chain is running like this NH C alpha CO NH C alpha CO it goes like that all the atoms are indicated here. So, now this is alpha carbon and then you have this is the peptide group, this is the peptide group. Now, these four atoms this is the carbonyl this is the double bond here and this is the NH here. Now, how many torsion angles are there here you have in between the dipeptide ring you have one torsion angle here another torsion angle here and a third torsion angle here. These ones are called as omega phi and psi. So, N C alpha torsion angle is called as phi and C alpha C dash or the carbonyl carbonyl is also called as C dash that rotation around this is called as the psi. Now, what does this rotation mean? Now, consider these four atoms consider the four atoms suppose I take this carboxyl carbon nitrogen and NH proton suppose I take this what will be the orientation of this bond with respect to this bond if they are exactly in the opposite direction if they are in the same side of these two atoms then we say it is this configuration if this carboxyl and this NH protons are on the same side of this bond typically one can write such kind of a things then we call this as a cis configuration if it is like this cis configuration. But if it is this is called as a trans configuration and the trans configuration meaning that this omega torsion angle which is indicated here this is 180 degree because that is the more stable structure there is a more stable structure and by and large that does not change at all why is that so because this carbonyl carbon this is a double bond. So this bond the N C alpha N C dash N C dash bond this also has a certain kind of a double bond character when there is a double bond character there is no free rotation possible here. So by and large the free rotation is restricted and there and the most stable structure is the one where you have this these two going in the opposite direction in the same way you can also say if these are in the opposite direction this car this C alpha and this C alpha will also be in opposite orientation. So therefore this is the peptide bond that we call therefore the peptide bond is a planar bond there is a planar the peptide plane is planar and now what about this now this is called as a phi torsion angle and this is the rotation around this bond. So which atoms do we consider? So in this just as we consider here the four atoms we consider these four atoms this carbon this nitrogen this carbon and this carbon if we consider these four carbons these four atoms what is the relative orientation of this carbon and this carbon with respect to this bond. So if they are in the same orientation same direction then it is a cis configuration or if they are in opposite sense this is called as a trans configuration. Now all in between things are also possible because this is the free rotation possible here because this is not a double bond here therefore there is a free rotation possible. So this determines the relative orientations of this amino acid with respect to this amino acid. So this is one torsion angle so and then the second torsion angle is this is called as the psi and that is a free this is also a single bond this is C alpha C dash C alpha carbonyl this is also a single bond. Now we can look at what are their relative positions of if you look at this four atoms nitrogen C alpha carbonyl C dash and this nitrogen if you take these four atoms with respect to these two this bond whether this nitrogen and this nitrogen are in the same direction or in the opposite direction the same direction will be the cis configuration other one will be the trans configuration you can have all values in between. So then we say the Gauch confirmation, trans confirmation typically we know from the stereochemistry these are the different possibilities so you have different kinds of possibilities here. So in principle they can all go from 0 to 360 or we also sometimes call it as plus 180 to minus 180 conventionally one can use both conventions whether it you want to define from minus 180 to plus 180 or 0 to 360 both kinds of conventions are used and initially I want us to figure out which are the possibilities. Now what determines these choices? Of course the steric contacts is an important factor there when you consider all these various rotations or all the orientations possible some of them are not because as a result of when you do this there can be clashes in the various atom the side chains of one amino acid residue can clash with the side chains of the other amino acid residue in which case that kind of a combination of the torsion angle will not be possible. So this is what was investigated in great detail by J N Ramachandran and there he calculated the energies of this dipeptide considering the possible different possible orientations different possible values of phi and psi and generated the values as the two dimensional map energy map he generated and what is shown are these the combinations which are possible and not possible. On one side you have the phi torsion angle on the other side you have the psi torsion angle and then considering the energies he said below a certain energy it is allowed below and above a certain energy is not allowed if that is because if you have a phi kilocalorie per mole if you have that kind of an energy you look at the Boltzmann statistics then you will find that phi kilocalories and above it is almost impossible to get any probability for that sort of a structure. Therefore he eliminated all those combinations which lead to the steric clashes and this is popularly known as the Ramachandran plot Ramachandran plot and you see all these empty space which is here this is disallowed this is disallowed only the red ones which are indicated here these are the areas where you can have a combination of phi and psi these are called phi psi maps or Ramachandran plot and these are the various values which are indicated here you have the 00 in the middle here and then you have the plus 180 and minus 180 that is the convention that is used and of course that is also the trans and here you have the Gauss conformations there. Now you see if your phi and psi angles are in this domain in this area this will lead to the formation of a helix structure that we talked about the helical structure this leads to the formation of the helical structure in that also there is a fine distinction made there is a so called alpha helix or the 310 helix in this one in the alpha helix there are 3.6 residues per turn in the 310 helix this is a 3 residues per turn this is somewhat more compact and you have that kind of a structure also possible and that appears above this strictly outside this range of the alpha helix. So phi psi torsion angles are indicated for the helical region on the other hand on this area this is a much larger area allowed area so you have the beta conformations there in this area you have the parallel beta sheet if you have these values here in the parallel beta sheet and you have in this area you have the anti-parallel beta sheet and there are also certain other kinds of helix these are more like it is called a polypropole if you have a sequence which has only proliens along then it actually it adopts the structure which is similar to the beta sheets although it is forms a helix but the combinations of the torsion angles are in this manner this is polypropole helix is more like a beta structure. So therefore this comes in this area then you also have what is called as the collision helix collision was the structure which J. N. Raman Chandran was working he was actually actually this came out as a result of his investigation on the collision structure the collision forms a triple standard helical structure. So it has very specific very special combinations of torsion angles and you have three strands there intertwined and that leads to a stable structure and that results in a helical structure but the torsion angles there are in this area therefore this is called as a collision helix. So you have the anti-parallel beta sheet the parallel beta sheet and then you have also a type 2 beta turn so this is a type 2 beta turn type 1 beta turn also appears somewhere here so you have these various kinds of beta turns which are appearing the secondary structures the combinations are possible. These are slightly extended areas these the thick ones are the ones which are very strongly favored and the ones which are slightly thinner the shaded these are less favored and therefore but they are possible it is not that they are not possible they are possible but they are less favored the thick areas are the most favored and then you see here you have a so called left handed helix what is present here is the left handed helix and this particular region which is alpha helix but it is a left handed helix these ones are right handed alpha helix this is the left handed alpha helix and you see most important thing is much of this two dimensional space is disallowed you cannot have combinations of this if anybody determines the structure of a protein with this phi psi torsion angles falling in this area then the structure is not acceptable because it will lead to clashes and steric clashes will be there and there certainly will not be a stable structure. Now with having looked at the various possibilities of the structures and we want to go into the NMR application how do you determine the structures and what are the characteristics of the protein spectra depending upon what is the nature of the structure and here is the typical illustration of if you have a protein which is very well folded that means it has the regular combinations of helices sheets turns etc everything present then you may have a protein the spectrum the proton spectrum this is the proton spectrum goes from 0 ppm to 11 ppm here is a beautifully spread out peaks here and in the case if the protein is unfolded in the sense that there is no particular preference for a particular combinations then there will be dynamics in the protein chain and there are multiple combinations are possible for each amino acid and then what we will see is an average of this chemical shifts. So for the individual amino acid the various protons which we talked about where which are present on the amino acids they will all be in a very narrow region here. So such a kind of a structure is called as an unfolded structure it has no specific combination no stable helix or beta sheet and things like that. So they will all be exchanging very rapidly and you will have an average chemical shift and all of them will form in this area. What are these different regions? So let us look at that and because of this different chemical shifts the NMR provides you a tool for determining the structures of the three dimensional structures of the protein. Now here is a detailed analysis of what sort of chemical shifts are present for the different for the different protons in your polypeptide chain. So typically this area from here to here these are the backbone amide protons this is almost from 6.5 ppm all the way up to 11 10.5 11 ppm you have the backbone amides and here you have the aromatic protons that is up to 8 ppm typically about 6.5 to or 7 6.8 6.9 up to 8 ppm you have the aromatic protons. Then you have the side chain amide groups where are the side chains these are asparagine asparagine and glutamines and also in arginine and lysine these are the four residues which had amide groups or the NH groups in the side chain and they will appear in this area from 6 ppm to 8 ppm. And then along the backbone you have the alpha protons this is as I said C alpha C alpha attached to the C alpha is the H alpha proton and then you have all other side chains here in the aliphatic groups and the methyl groups here. The methyl are there for the valines, isoleucine, alanines and then the threonine so all of these methyl groups appear in this area. So therefore they are very characteristic chemical patterns therefore for the different types of protons in your polypeptide chain. Now here I want to show you the once again the same amino acids but the nomenclatures of the individual atoms. So this is individual residues you notice here where is the chain going this is the carboxyl and this is the nitrogen and these are now in the amino acid in the polypeptide chain as they are present in the polypeptide chain not individual amino acids as they are present in the polypeptide chain. So this is the nitrogen then the C alpha carboxyl this is the chain which is running like this and then the side chain is going like this you have the C beta here the C beta this is the methylene group so this is H beta 1 and H beta 2, H beta 3 here there are 2 and then you have the C gamma. So then you have this C gamma has again 2 protons there H gamma and then you have the C delta once again they have this 2 protons there. And this joins the nitrogen therefore proline does not have a free amide proton on the backbone on the in the glycine for example has 2 alpha protons here alpha protons in the chain NH, C alpha, C orange like this alanine has 1 methyl and 1 alpha proton arginine has there is 2 beta protons 2 gamma protons 2 delta protons and then from this epsilon you have this 1 amide proton here and then you have this another carbon going over to the NH 2 group here then the N and then NH and NH 2 there are so therefore arginine is a quite a elongated chain so quite a bit of basic nature here because of this so many nitrogen present here. And as per gene as we said already had CO NH 2 and the CO NH 2 is this you have the CO H CO OH here and then you have this NH 2 there this is the reminculation called as delta these atom names are labeled as gamma and delta and so on and so on. And as partic acid again has the CO OH here in the side chain C beta at the gamma position you have the CO OH and 16 as the gamma position you have the SH and then you have the 2 beta protons there and glutamine has again the CO NH 2 with the gamma proton has 2 with the gamma position you have 2 protons and this one similarly in the glutamic acid you have CO OH in the side chain here and that appears as the delta why I am going through all of this is because these one nomenclatures one will use very frequently when you describe the protein structures we say here is the proton beta chemical shift gamma chemical shift delta chemical shift and so forth. Therefore one should know which proton we are talking about in the backbone and histidine has this nomenclature here you have the n epsilon there is a proton here and then there is a proton there which is attached to these are the 2 protons which are there is an exchange here which can happen between these 2 proton. So similarly we have this alpha beta gamma and delta for all the amino acids it follows in this is a typical convention which is followed in the IUPAC nomenclature of the amino acids. So you immediately you can see here now whatever what we are trying to show here now these are the various amino acids alanine, cysteine with the 20 different amino acids and what are their proton chemical shift ranges the proton chemical shift ranges you see they are quite distinct although there are certain range of overlaps here these are many things which are overlapping in this area but and the alphas are appearing in this area which you already indicated there and these ranges indicate the variations possible depending upon the protein structure what is the kind of an environment for this particular alpha group. So depending upon that you have certain variations. Now the distinctive features are of the alanine okay so the beta is very distinct here okay so with regard there is only one alpha and then there is a beta and the cysteine has the beta which is somewhere here as part it also has a beta here and glutamate has the gamma and the beta so you must have you must be able to connect all of those ones there the felony alanine is alpha and the beta of course there is no connection to the aromatic rings here aromatic proton positions are not shown here because they will also be aromatic ring protons there the glycine is only alphas histidine is alpha beta so these are called ABX spin systems okay all these are long chain side chain these are called long side chain spin systems we have this and then you have some which are only alphas and the betas there are many reserves which are only alphas and betas see this one is alpha beta and of course this one is alpha beta this is alpha beta and this is of alpha beta gamma alpha beta alpha beta okay there are many like that okay this one is an alpha beta okay and once again this is alpha beta but the serial beta is very close to the alpha area this is because of the OH group OH group which is present on the side chain the beta proton comes very close to the alpha there and tryptophan these again alpha beta tyrosine is alpha beta and the threonine also has alpha beta which is very close to the these are very close because once again there is a OH group on the threonine side chain but it has also a methyl so that is a gamma and that distinguishes it from the serines threonines and serines are distinguished because of this although these areas are overlapping but the threonine has a methyl in the gamma position and the valines alpha beta gamma now here are there is an experimental spectrum and this is a so-called toxic spectrum and you can see here the various amino acids which are typical peak patterns what you get and this is essentially I am what is listed here is explicitly shown in this here you have the alpha beta which alpha betas will be present all alphas will be present but the betas of the serines and threonines will be there but of course in this sequence there is no threonine you only have the serine there okay all alpha are there for a particular section of a protein spectrum okay and then on this side you have there the the betas and the gammas and all of those and this area is belongs to the lysines okay and these are the side chains this area belongs to the side chains these appear in pairs because the CO NH2 the two protons on the NH2 are non-equivalent and they will be connected to the thing in the side chain in this manner so they will be in pairs they will appear together okay and so these from each of the backbone amide from the each of the backbone amide so you see the correlations you can see this piece speaks in the toxic or you will also see them in the nosy in the nosy spectrum we already talked about the toxic and the nosy I will show you again those ones below and here you have the correlation the glycine so the glycine is here and you look at the serine here serine alpha is here and the beta is here which is quite close there is one glycine here another glycine there there is no other one below or above that okay so you have therefore the lines are drawn here to indicate this they all belong to a particular amino acid and from that particular amino acid you have the same pattern okay so you look at the lysine here this is the alpha of the lysine and goes all the way to this beta gamma and all of those ones they are okay so now here from histidine histidine you are seeing to this beta and it does not have any other one there and the side chains these are epsilon okay from the side chains of this once you will also see to the betas of the asparagine and the glutamines and also sometimes you will see to the epsilon's of the lysine's arginine's and lysine they will also appear in this area and they will see those ones are indicated here you can see at the lysine see the beta gamma and delta they are appearing here and the epsilon is appearing here the epsilon is at chemical shift for 3 ppm very distinct okay and sometimes you may see these peaks you may not see this peak you will see them in the noosey but you may not see them in the toxic spectrum because it has the relay has to go all the way down depend upon the what is the mixing time used in the toxic spectrum it may not reach up to the epsilon but it will certainly reach up to the alpha beta gamma and sometimes to the delta so you will see distinctive intensities for this different peaks as you are looking at that of course what is present here is the water okay okay now this is a typical cozy spectrum of a peptide and the various regions are indicated there so what are these here these ones are the aromatic protons as we said the aromatic ring protons have correlations among themselves the coupling is between themselves so in the cozy spectrum you will only see cross peaks here for the aromatic ring protons and then from these ones and this area belongs to the NH C alpha H NH C alpha H NH proton is coupled to the C alpha this is a three bond coupling and you will see those ones in this area so therefore the cozy spectrum so the NH to the C alpha H now from the C alpha H of course you will see to the C betas here and the beta to the gammas you will see beta beta etc this of course region is a quite a crowded region there so one can identify only from the alpha to the betas here then you have the beta gammas and then you have the gamma deltas okay and delta to the epsilon and the methyl etc they will all come in this area so this is typically categorized in certain boxes here depending upon what sort of a proton pairs are involved in this generation of the cross peaks okay so you see here the difference between the cozy and the toxic and this is the same this is again the NH to the alpha area we saw earlier so NH to the alpha this appears in this area from 9 people around this area 7 ppm to the 9 ppm you see a cross peak to the individual alpha amino acids alpha proton of the same residue in this case a particular region is taken you have this 5 peaks there of course they have a fine structure which is expected because all these protons have a fine structure and that will reflect in the cross peak also therefore you have 1 2 3 4 5 you can identify this 5 amino acids there and then corresponding to those ones of course you will also in the toxic spectrum you will see additional peaks and what those additional peaks are they will belong to the beta the gamma and the delta therefore looking at this kind of a pattern what you will have you can identify what sort of a spin system what sort of an amino acid it is for example this one has many protons which are connected to it right so therefore this clearly a long side chain and this one is has only one fellow here so this is alpha and then there is only one there are two peaks here possibly there and these ones are the beta 1 and beta 2 so the relay has happened from the alpha proton to the beta 1 and beta 2 there so and now this one has now this amino acid has an alpha proton here and then it goes to the to the beta here and then to the gamma there so therefore this also is a little longer side chain see now it see it goes into the methyl area once it comes into this area it is the methyl proton region therefore these are likely to be like the valines and the isoleucines or leucines and things like that okay now you look at the next one here so this alpha proton has two peaks here and these are around the 3 ppm and looking at the table water showed you here this is again a 2 beta protons of a particular amino acid residue and these are most likely to be the aromatic ones aromatic ones are present in this area this is the 3 ppm these are the tryptophan, phenylalanine, tyrosine and sometimes the histidine so you might find these it can belong to this kind of residues and now you are here you have the one of the last alpha proton there now this peak corresponds to this here okay but this one also has a certain other ones residues here which are going like this so therefore there are threonines or the beta protons or the alpha protons and so and so forth that can be some other residues it can be overlapping over there this is what you see in the toxin and this is the more detailed region or the other region of this of the same spectra so you have coming from in the alpha in the aliphatic area the aliphatic area you have this various correlations the beta 1 and beta 2 alpha to the betas and this will be the alpha to the beta cross peaks then you have to the beta to the gamma cross peaks then you are gamma gamma cross peaks here so you will have a whole set of cross peaks which one needs to analyze looking at the sequence what you have then you can fix those okay so now that is for the identification of the individual amino acid residues now you have to connect them sequentially cosy spectrum only shows within the same amino acid residue cosy does not show peaks between amino acid residues one residue to another residue it does not show because there is no such proton-proton coupling which can tell you that okay however these ones will appear in the nosy spectrum because the nosy spectrum reflects on the proton-proton distances okay so the proton-proton distances are can be quite varied depending upon the structure what you might have in the protein so you certainly have near neighbor interactions near neighbor interactions are the sequential interactions you can also have a long range interactions which will determine the secondary structure of the protein okay so there is a brief of the distances here the various distances which are present in the polypeptide secondary structures. Let us look at this particular we will have to go through this again in a greater detail and the polypeptide chain is indicated here NHC alpha CO NHC alpha CO NHC alpha CO it chain is running like this residue I residue I plus 1 I plus 2 I plus 3 and so on so forth. The why you are taken 4 residues because we are having we are going to have short distances between the protons as far as 4 residues away and that is in the regular secondary structures therefore one has to show these 4 residues and it is the various short distances that indicated by lines there and we will go through these individual ones possibly in the next class I will stop here.