 We will now start a new application of NMR in structural biology that is with regard to the determination of the structures, interactions and dynamics of proteins. What are proteins? Proteins are polymers, they are built from amino acids, a typical structure of an amino acid is indicated here. So this is the NH2 here, then there is an atom called as a C-alpha carbon and to that is a raised to proton and then it is a R1 which is called a side chain and then it is a carboxylic acid group here. So this is the basic structure of an amino acid, you have the so called N terminal, it is an amino group and then you have the C-alpha one carbon atom here. So this of course you can have various different kinds of groups attached which are called the side chains and then this is called a C-terminal which is the carboxylic acid. Now two amino acids can join together in this particular manner as it is indicated here. The OH group of the carboxylic acid and this one hydrogen of the amino group they can get removed by the formation of a bond between this carbonyl carbon and the nitrogen here and that is how you see this bond is formed and a water molecule is removed. So this remains R1 and the another amino acid which is here amino acid 2 which has a side chain which is called as R2. So the R2 is here and you have formed a bond here with the CN bond, this is the N-terminal of one residue is now bonded to the C-terminal of the another residue and this is called as the peptide bond. So the chain continues like this so we have here the OH group once more then of course this carboxyl of this second residue and it can form peptide bond with another residue with the amino group of another residue so it will continue like this. So therefore this is indicated more schematically in this so you have this COOH group here and the amino group here this hydrogen and this OH group are combined to form a water molecule and then there is a bond carbon nitrogen bond formed here. So therefore you have this CR and then you have this carbonyl, carbonyl 10 to the NH group so this is the peptide bond. So this group is called as the peptide group COOH and then you have the CHR there is a CHR these residues R, R can be the same or it can be different so therefore they can have different kinds of linkages between the various amino acids. Now there are 20 different types of amino acids what is the difference in them the difference is in the R group the difference is in the R group here for example this is called the L alanine, this is called as porazine and this is called cysteine and all these are L groups this depends on the configuration at the C alpha carbon. So which way the groups are oriented here the CH is indicated on the top R1 is indicated here so which way these are relatively oriented that determines whether it is L amino acid or the other thing is the D amino acid. So by and large in nature we only have the L amino acids where the proton is on a particular side and the hydro side chain is on the opposite side so all these are L amino acids. So these the residues which are indicated here so we are group them into different categories so you have the L alanine, L asporazine, L cysteine, L glutamine and then you have the glycine, the isoleucine, lucine and methionine. Then you have the phenylalanine then you have the proline, you have the serine and threonine. So all these amino acids are so called neutral amino acids neutral in the sense that after the peptide bond is formed they do not have any charge anywhere. So there is no protonation site so after the peptide bond is formed in the polypeptide chain when you form a chain like this so what is the nature of these residues? The alanine has the side chain is a methyl group and then in this asporazine you have the CO NH2 group these are all neutral although they are polar there is some polarity there it has a CO NH2 group and then the cysteine has an SH group here the side chain H has an SH group. We call this as the after the C alpha you have the C beta and then the SH group so this is the C beta carbon from the C beta carbon we see the nomenclature afterwards and the glutamine has this carbon here another carbon then you have the CO NH2 group. So there is a common thing between this glutamine and asporazine they both have the CO NH2 so there is a CO and the NH2 in between there is another carbon in the case of glutamine whereas that is not there in the asporazine. And then you have the glycine, glycine does not have anything else it only has two protons at the C alpha position there are two protons so this carbon here has two hydrogen therefore there is the CH2 group there and isoleucine isoleucine has from here three carbon branching out like this there is a proton here there is a carbon here and that carbon is actually methyl group that is a methyl group all the protons are not drawn here only the carbons are drawn. So there is a carbon here and a carbon there the structure there is a branching at this site at the C alpha there is a branching here is another carbon here and one carbon here and one carbon here. So obviously from here it is clear that if you have a sticking a stick just staying by itself then it would mean that this carbon will be a CH3 group because it is a S3 hybridized group there are no other bonds indicated it is only protons attached therefore this will be a CH3 group. So this are the CH3 group here and then this will be a methylene group the CH2 and then at the end of course you have another CH3. So the end if it is there is no other branching it is this it will be CH3 and the leucine and the leucine has from the C alpha this is the C alpha to the C beta and then the C gamma and there are two methyl there because you see the two sticking around here these are two methyl groups because their bonds are drawn only to the carbon carbon bonds are drawn. So this will be a methyl group there are two methyl groups there and then for the methionine methionine it goes in this manner from the C beta so you have an SA there is an S here but now S is connected not to a proton but to a methyl there is a CH3 unlike the cysteine the cysteine had an SH here now this does not have an SH but it has a CH3 and it also has a methylene in between so there is a methylene group with the CH2 and then the SH3. Now these are aromatic this is phenylalanine you have the NH2 from the C alpha you have to the C beta and to the C beta is attached to a phenyl ring so this is a phenyl ring and now look at proline is an interesting structure and this is this forms itself within on the backbone itself formed a closed ring all these are CH2 CH2 CH2 NH one of the H of the proline is the one which is normally remaining it actually participate in the closed ring formation therefore in this hydrogen bond is formed and of course this proton will also go and there will be no other proton remaining on the proline ring. So therefore this the proline ring in the polypeptide chain will not have any hydrogen at the nitrogen position. And the serine now has a CH2 OH group in the side chain the CH2 OH so this is the CH2 here and then there is the OH there from the C alpha there is the CH2 the CH2 will call as the beta proton and the threonine is similar to the serine except that you have a methyl there is no proton there so there is instead of the proton there is a methyl group here in the case of serine there is a CH2 and then the OH now here you have the C and then there is of course a proton there one proton and then there is a CH3 group as attached to this. So this is the these are the different structures of the amino acids now all these are neutral amino acids once the polypeptide chain is formed there is no charge anywhere on these ones. Now so here we have the next set of amino acids these are called as acidic amino acids and these are here L tryptophan, tryptophan, tyrosine and valine so these are also neutral because there is in the color coded manner you can see tryptophan has an indole ring here at the C beta at the C beta attached to an indole ring so we have this is the tryptophan and the tyrosine is very similar to phenylalanine except that it has an OH group here. So all these amino acids so therefore 4, 4, 8 plus 4, 12 plus 3, 15 amino acids are neutral in nature and then you have these white ones this is the acidic, acidic there are 2 there so these are aspartic acid and glutamic acid why because in the side chain you have a COOH group and the COOH group is acidic you have the CH2 then you have the COOH the backbone COOH is different it is the backbone COOH which participates in the polypeptide chain formation but on the side chain you also have a COOH group therefore there is chance of having a negative charge there because this will carboxylic acid group is there that will OH group will dissociate so you will form a negative charge there so you can have a negative charge in this these are called as aspartic acid this is aspartic acid and it has it will be acidic similarly the glutamic acid glutamic acid is similar to this except that it has 1 more carbon in this here there is a CH2 CH2 and then COOH so these 2 are acidic amino acids then you have the basic amino acids there are 3 basic amino acids the arginine histidine and lysine so these ones have NH2 groups or NHNH2 groups in the side chain so you have the alpha C beta then you have the gamma then you have this delta then you have the epsilon so you see this goes to the epsilon and xi so this chain is quite long these side chains are very long so there is dephrode there are many carbons here in the side chain similarly the histidine histidine has a 5 member ring in here so there is a 5 member ring there are 2 NH2 nitrogens and one of them has a hydrogen there so this also can get protonated because of the nitrogen they can get protonated then when it gets protonated it forms a NH3 or wherever the charge is there it will produce is a positive charge the acidic one produces a negative charge the basic ones produce a positive charge and this is the lysine the lysine has so many carbons on the side chain this is the C beta from the C beta you have the gamma delta epsilon zeta then you have the NH2 group there so all of these are very distinctly different amino acids typically all these are denoted by 1 letter codes you can have the 3 letter codes as well as the 1 letter codes alanine, asparagine, cysteine, glutamine and these are represented as A, N, C, Q like that gly, I, L, M and then you have F, P, F is phenylalanine and you have the P is peroline, S is serine, T is threonine then your W is tryptophan and Y is tyrosine, V is valine. Valine also has 2 methyl groups here there is a C beta and directly to that is the 2 methyl groups in this and arginine so this is R represented as R then histidine and K. So this is the structure of the various 20 different amino acids therefore clearly the polypeptide chain can have a great variety of sequences and depending upon the combination what is the nature how long is the polypeptide chain and which amino acid is appearing where how the structure is getting formed and that determines the first level of the structure of the proteins and that is what is indicated here as the primary structure of the protein and these are like beads, these are like a beads on a necklace so each one of these beads is amino acid these are all amino acids there can be many amino acids they can have proteins which have 100 amino acids, 200 amino acids, 1000 amino acids so various kinds of amino acids are present. Now each of these amino acids has a certain degree of degrees of freedom with respect to how they are oriented with respect to each other. So in the entire chain there are many different possibilities of orientations of the individual amino acid side chains with respect to the previous one and the following one. So depending upon you get some regular structures and these regular structures will show up when you have a large chain like that going with certain preferences for the certain relative orientations of the individual side chains along the backbone then you get different kinds of structures. These are classified in a rough way like this. This is called as the alpha helix it goes in a helical manner and these are called as the beta sheet it looks like this we will look into this in greater detail soon and this is a kind these are called as secondary structures this is called as the primary structure and this is the secondary structure you have two kinds of basic secondary structures alpha helix and beta sheets there are two types of beta sheets that we will see very soon and then these ones relative orientations of these secondary structures in a protein the protein can have partially some places it can have this structure some places it can have this structure so therefore we can have a combination of all these secondary structures in a large protein and that is called as a tertiary structure the tertiary structure basically represents the three-dimensional structure of the protein so you see you can have the beta sheets or you can have a helix here the helix is indicated in this manner here you can have many helices here then you know beta sheets combination of all of these will form the tertiary structure of course the chain can loop here and then those ones will be called as as the turns in a particular molecule there can be combinations of alphas and the beta sheets and there can be connections in between and those are called as the loops there can be turns there these are also called as turn there are specific geometry of the relative orientations of the individual amino acids then you have the cotonary structure you have a well-folded structure like this then can multiple domains there can be more of such ones we can get associated because of various interactions and these are called as cotonary structure therefore the protein structures can be categorized into four different types so you have the primary structure which is the basic thing which is which determines the composition of the protein what all amino acids are there whether it is the basic protein or it is an acidic protein or it is a neutral protein or it is a hydrophobic protein because certain amino acids you see as we have seen have various methyl groups aromatic rings they all constitute to the structure the physical characteristics of the amino acids the physical characteristics of the amino acids pass on their nature to the entire structure of the protein because of that they also define the energetics and then they cannot different orientations and then you will have a different kinds of folded structures and then these can get associated through various kinds of interactions they may be charged charge interactions or hydrophobic associations different kinds of associations can happen and that will constitute the so-called cotonary structure. So let us look at this in somewhat more detail so we have here the primary structure is indicated there so we have different kinds of chains which is the so-called carboxyl end this is the end terminal the chain is running like this of course it has various loops going around and different kinds of interactions can happen there so and then we show here what is the nature of the helix in little bit more detail if you see the helix the chain is running the end terminal is on this side say NH C alpha CO NH C alpha CO NH C alpha CO and that is the end terminal so it goes from the end terminal to the C terminal like this CO NH so now you see there are why is it taking this sort of a structure it is taking this sort of a structure because this is stabilized by what are called as the hydrogen bonds so you look at here these are the hydrogen bonds the NH of the of this amino acid is hydrogen bonding to the carboxyl oxygen of this this is the NH group here and there is a hydrogen bond between the carboxyl group of this this amino acid this comes at a certain distance after it is taken a certain turn here the helix this forms a helical hydrogen bond this provides a stability to it likewise every amino acid this this carboxyl group will be hydrogen bonded to the next of somewhere there and similarly so this next amino acid here is hydrogen bonded to the carboxyl of here so this continues so every in this whole chain every amino acid amide hydrogen is hydrogen bonded to the carboxyl of one of the of the residues in the polypeptide chain so this is called as a turn of the helix so this is the turn of the helix and it continues like this and in the turn the total rise of this is typically about 3.66 angstroms 3 there are 3.6 a turn amino acids in a turn 3.6 amino acids in a turn so there can be different kinds of turns there are called as 3 amino acid turns they are called 3 tanylases and that determines how many what is the size of this ring if you consider this as a total ring what is the size of the ring can define the size of the ring there then you have the how are the beta sheets formed here you see this is the within the same chain a fourth residue is actually forming hydrogen bond with the amide group of this and that forms the hydrogen bonding scheme now in the beta sheet this is it is slightly different okay now look at what is this chain how it is running here so the chain is running NH C alpha CO NH C alpha CO NH C alpha CO so this is the chain running like this this is from the N terminal to the C terminal now the lower chain is actually going in the opposite direction here NH C alpha CO NH C alpha CO NH C alpha CO and so on so the chain is running in this manner here therefore this is called as an anti-parallel beta sheet there are two chains which are running in opposite orientations and then these are held together by this hydrogen bonds this NH is hydrogen bonded to the carboxyl of residue in the other strand this is one strand this is the other strand and the two strands are held together by hydrogen bonds like this so and they are perfectly positioned to form this sort of hydrogen bonds NH CO and then the NH CO here like that it continues so there are very regular intervals this many hydrogen bonds are possible this forms the one of the beta sheet this is called as the anti-parallel beta sheet okay there can be others also so the tertiary structure here this is the helical portion is shown in this this is the helix okay the arrow comes here from here you see it comes and points to the helix here this is the helix and this portion is pointing to this here so it is turns here therefore when it turns like this the change in the direction okay one is going on from here to here then it goes from here to here so there is a change in the direction and therefore this will form a beta sheet the same thing is indicated here this portion of the chain is supposed to be forming a kind of helix whereas this portion of the chain is actually there are chain is turning here and then of course you will form hydrogen bonds in this and that is typically indicated in this sort of a structure okay now you see there are certain things which are hanging here what are these because the chain has to fold and even here if the chain has to turn here then something is here what is this what does this correspond to these are called as turns okay now there are two kinds of turns so the chain goes in this manner which way the chain is running we can look at this so this is the C alpha CO NH C alpha CO NH C alpha CO NH C alpha CO so there are four amino acids here one two three and four these four amino acids are forming a turn and the carboxyl of the first amino acid for carboxyl of the first amino acid residue is hydrogen bonded to the NH of the fourth amino acid residue see it is turns like this and comes back here and in this situation this NH is coming close to this carboxyl it forms a turn notice here the carboxyl of this the secondary residue is pointing inside here and the NH is pointing outside this is one configuration this depends upon as I said depends upon the relative orientations which are written by certain certain kinds of torsion angles I will describe a little bit later very soon and in this case this is called as the type 1 beta turn this is called as the beta turn and this is called as a type 1 beta run there can be another way this turn can happen and in this it is the same hydrogen bond except that the configurations here is somewhat different you see here the R H product of the hydrox the alpha proton is down here whereas the alpha proton was up there and the two things are in the same orientation and the carboxyl is oriented outwards and there the carboxyl is oriented inwards and the NH is outwards the NH is inwards here similarly this R 3 and this portion is roughly the same as that it is the difference is occurring in this so and this amino acid residue has a different or relative orientation with respect to the others and that makes the configuration in the loop different or we also call it as the conformations is different and because of that the same hydrogen bond is found even so here so this is called as the type 2 beta turn okay so now you see here this is more explicitly indicated what makes these different orientations and what is shown here is there are certain torsion angles indicated here this residue if this residue is I this is the residue I plus 1 this is the residue I plus 2 and this is the residue I plus 3 this is I the chain is running like this NH C alpha CO NH C alpha CO NH C alpha CO and continues like that so if I start this from this residue is I plus 1 this previous residue is I NH C alpha CO this is the residue I and this is NH C alpha CO this is the residue I plus 1 and there are two torsion angles indicated here this actually determine what is the relative orientations of these groups these torsion angle they are called as Phi and Psi we will describe more about it these are called as the torsion angles Phi and Psi similarly this amino acid also has the Phi and Psi torsion angles and this define the relative orientations of these groups these four items is nitrogen this C alpha CO and this nitrogen what are the relative orientations of this a rotation around this can change the orientation of this position of this hydrogen and with respect to this and that is what determines all of these so like therefore these two torsion angles which are called as the Phi and Psi these are there for every amino acid residue and that determines the variety of structures that can be formed by different combinations of this Phi and Psi torsion angles and when this turn the chain turns around and comes back here you see this distance is roughly about 7 angstroms okay so one can calculate this various kinds of structures depending upon what is the configurations of this if the hydrogen bond has to be formed this hydrogen bond has to be formed this puts the limit as to how much should be the distance because from N to O this distance cannot be more than 2.9 angstrom about 3 angstroms 2.8 to 3 angstrom this nitrogen to this oxygen will be about 2.8 to 3 angstrom that defines the hydrogen bond and once we have that this is the c alpha c alpha distance this is the c alpha carbon this is the c alpha carbon this distance is approximately 7 angstroms okay now we talked about the antiparallel beta sheet this is the antiparallel beta sheet there is one more possibility of a beta sheet and that is called as the parallel beta sheet how does this work now you see you consider these two strands this we already saw consider these two strands N H C alpha C O N H C alpha C O N H C alpha C O the chain is running like this from left to right and here also N H C alpha C O N H C alpha C O this chain is also running in the same direction whereas here the two are running in the opposite directions here it was running N H C alpha it is going here the bottom one is going in this direction and here the two chains are running in the same direction and yet of course the hydrogen bonds are possible. This NH now hydrogen bonds to this oxygen. Here the hydrogen bond is scheme is much more cleaner, they are straight and of course then it is more stable and here of course they are slightly tilted there. So this NH is hydrogen bonded to this oxygen, this NH is hydrogen bonded to this oxygen. So this will continue like that alternately like here this NH to this CO, this NH to this CO, this NH to this CO, this NH to this CO, this NH to this CO. And similarly here there is one, there is a shift in the resistor. So therefore you have this CO to this NH, this CO to this NH and this will of course go further down. This is called as the parallel beta sheet. Now in a given protein structure you can have combinations of these. The chains can be running in particular directions. Here the chain is running like this NH, CO, CO, NH, CO the chain is running like this and it may have a turn somewhere and comes back and then another portion of the chain runs like this. So this is NH, CO, CO, NH, CO. Therefore here it forms a parallel stranded, anti-parallel beta sheet, anti-parallels beta sheet. This portion is anti-parallel beta sheet. But again the chain may turn around somewhere and comes back and it goes once more here NH, CO, CO, NH, CO. Now this direction is anti-parallel to this orientation the middle one. It can again form hydrogen bonds. Therefore this is once again an anti-parallel beta sheet. So these two anti-parallel beta sheets are adjacent to one another but then of course this chain may go somewhere and then again you may have another chain which is running parallel to this NH, CO also for CO, NH, CO, CO and so on. Now this chain is running parallel to this. So therefore this beta sheet which is formed here is the parallel beta sheet. So such kind of a sheets can be formed in protein structures. Depending upon the relative orientations of the different helices or the chains you can have anti-parallel beta sheets and parallel beta sheets you can have combinations of this. It can form a huge sheet like structure as a result of this and this will be extremely stable. Of course this will be determined by the side chains which are present. The side chains will dictate what sort of a structure that can be formed and that will depend upon as I said the torsion angles the phi and psi torsion angles and this of course is a very crucial parameter which generates a variety of protein structures. So I think we can stop here.