 Good morning. So we were discussing basics of solid state NMR spectroscopy and its application in a structural biology. So in previous class, previous two lectures we have discussed why solid state NMR, what kind of biological sample we can investigate, those biological samples that are not amenable for crystallography or neither they are soluble, they are perfect for solid state NMR, for an example membrane proteins, integral membrane proteins or amyloid fibres, these kind of difficult molecules that cannot be specialized neither they can be dissolved in solution for doing liquid state NMR, they are perfect sample for solid state NMR. We also looked at how magic angle spinning improves the resolution, then we discussed about cross polarization, how cross polarization increases the sensitivity. Briefly I touched upon decoupling that is required for again resolution enhancement and I said that we can use decoupling techniques for getting the lost structural information. Today I am going little more detail into these techniques and also I briefly touched upon proton detection, why it is needed and what can be done using proton detection. So let us move ahead little bit repeating what we discussed. So here is our sample packed in a rotor, this is the solid state NMR rotor and this rotor is placed at a particular angle that is called magic angle. This is the direction of magnetic field that is in z direction. Now this sample is spinning at very fast speed and that enhances the resolution. So essentially magic angle spinning averaged out the chemical shift anisotropy and it helps in getting good sensitivity and resolution. So what it does? So let us take an example of a simplest molecule like glycine. So this is a simplest biological system glycine with two carbons, one CHCO and another CH2. So we are getting two broad peak, one centered around 50 ppm another centered around 170 ppm. When we start spinning this anisotropic interactions that are because of different chemical shift that we discussed last time there, these spins are oriented in various direction and resultant gives you broad spectrum. So when we start spinning these are getting splitted and we are getting many line as we increase the spinning speed about 5 kHz you see we are getting two sharp line one centered around 170, one centered around 50 ppm. And at 14 kHz we are clearly getting two very sharp peaks. These corresponds to C over resonance, this corresponds to C alpha resonance. So that is what magic angle spinning does, it averaged out the chemical shift anisotropy and it enhances the resolution and sensitivity. Now how to spin and where to spin what we use for spinning? So as we discussed we have a different size of rotors. These rotors are made up of zirconium oxide and here we put the biological sample. So depending upon what is the size outer diameter of these rotors we can choose a spinning speed like here 7 mm or 4 mm, 4 mm can go up to 14 kHz in broker system, 3.2 mm can go up to 24 kHz, now 2.5 mm this is 1.3 mm. So depending upon what is the outer diameter this rotor can go up to various speed like this can go up to 65 kHz. We can really now using these people can detect proton and step of the art is 0.8 mm rotor that is used for proton detection it spins really fast about 120 kHz. So these are the rotors where we put our biological samples and these has to be robust because we are spinning very fast. Now for spinning we discussed that we are using air so there should be some caps here so you see the caps which has a fins here. Now here the drive pressure and bearing pressure that we are giving in the probe that helps in spinning and these floats and for detecting whether the rotor is sitting in the right slot or not we have here the black mark that basically using optical detection system it detects and helps in spinning of the rotor. So these are the caps that are used for packing the samples these are of different shape and size like this is there are two prominent one is made up of KELF and one is called waste pill. So these are two different caps that are used for packing the samples and spinning it. This rotor has to be really carefully packed it should be balanced it should not be leaking and caps should be tightly fitting otherwise it can break. So really one has to be careful by packing the samples and spinning and for solid materials there are some tools that is provided by the manufacturers that basically these tools helps packing the samples inside the rotors. Now where does it sit so as I said magnetic field is in z direction here is our probe dissection of the probe here our rotors these rotors are sitting. So depending upon what size rotor is the whole dimension is accordingly fitting it. So this is this whole assembly is called a stator that is oriented at a magic angle and here we have a flexible coil connections they are these are various coil for detection and also sending the pulse here is the place where rotor actually spins using air that comes from bottom. So there are two main air bearing air and drive that actually spins here in the stator and that is create the different spinning speed that we were talking. So the another term that we had discussed is like a decoupling that in improves the resolution. So decoupling is what we say we are this is heteronuclear decoupling. So we are exciting carbon with 90 degree pulse and detecting carbon this is one dimensionless spectrum so S spin is being detected after the irradiation with a 90 degree pulse. During that detection period we are applying a high power decoupling that decouples the proton carbon coupling that we had seen that it loosen out the interactions. So that improves the resolution. So here I just in a schematic I have shown you if I have a static spectrum we get really really broad peak when we decouple we improve little bit of resolution but with mass we increase improve quite a bit of resolution and this is adamantine which has two flexible carbon and now with mass and decoupling we can really get two sharp peak with increase in resolution as well as sensitivity. So to re-emphasize this magic angle spinning with mass and decoupling these are two basic building block for any solid state NMR experiment that improves the resolution as well as sensitivity. So this is kind of like a CP pulse sequence and dipolar decoupling that I show you. So just to have an analogy this is our 90 degree pulse in liquid state we are exciting the magnetization using 90 degree pulse in liquid state and then immediately we detect it right. But in general case in solid proton dipolar coupling is huge we cannot detect it when we are spinning at moderate speed. So for that purpose we detect mostly on the X nuclei which is carbon. So carbon sensitivity you know gamma is 1 by 4 so therefore its sensitivity is going to be low. So what we do we do something called cross polarization that we had discussed so we excite with proton and by matching this condition which is called Hartmann hand matching condition we transfer that polarization to carbon we detect on carbon while decoupling the proton. So this pulse sequence is called CP sequence cross polarization and the duration for which we are cross polarizing is called T contact time contact time is here TCT the duration for which we are cross polarizing it. So you know this has to match that Hartmann hand condition omega s gamma s equal to omega i gamma i that is condition has to match for cross polarization to happen. So this again improves the sensitivity of the solid state NMR experiment. So we are just I am showing it what is happening so let us take a mixture of two solids. Now if we do direct polarization means direct polarization means just applying a 90 degree pulse here on carbon if you just apply a 90 degree pulse on carbon and detect it that is called direct polarization like here what we do in liquid state. If you do direct polarization what we are getting a broad spectrum with no resolution. But when we do cross polarization now you can see all these peaks here alpha beta in this mixture of a compound where one is negatively charged another is positively charged these two are mixed to make a kind of jelly kind of substance and if you do cross polarization we can enhance the resolution as well as sensitivity and the enhancement in signal can be directly proportional to the ratio of their gyromagnetic constant. So this is this what we achieve by doing cross polarization. So Cp and mass are building block of any solid state NMR spectrum. We achieve the sharp signal that we had discussed earlier that is needed. Needed for getting any any information that is there. But we are we have paid a huge price by achieving this spectral resolution we lost all those important interactions that were present in the solid inherently like chemical shift in isotropy quadruple or coupling dipolar coupling and J coupling by doing this mass and decoupling we have lost it. And this is required for a structural information so somehow we have to get it back right. So this is something called you have a cake and eat it. So we want to have a cake and eat it too for doing that we need to do some more trick decoupling is not enough mass is not enough we have to do something called a recoupling we have to recouple so that the lost important interactions are brought it back. So that is what we do we had lots of interaction lots of interaction present inherently because of the spins were static now we force them to to rotate by mass and then by decoupling. So decoupling all these spins become individual. Now we want to selectively introduce the interaction between them that is called recoupling. So here we are recoupling it now by applying some trick either during spinning or applying some pulse we need to recouple so that our interactions are brought back and this technique is called decoupling by application of various RF very couple it. So decoupling is breaking the interactions breaking the interaction because of CSA and DD that were present inherently and decoupling is selectively reintroducing these interactions that is what a recoupling means. Now we can selectively know that which interactions is happening with which protons so that is that is what recoupling means. So how it is done it is very simple I have to do some trick one of the important or essential trick is like can we do something with because there are three factors one is RF that is given by us one is already we are introducing the spins. So suppose we have a proton we have a two peaks here one for C alpha one for C o. This is about 50 ppm and this is about 170 ppm. So what is the difference between these two is 120 ppm and suppose we are doing this experiment at say 600 megahertz. So let us see what in simplistic term what we can do. So let us take our glycine right glycine one we have a peak coming from CH2 one coming from C o here are that is what we saw one around 50 ppm and one around say 170 ppm these are the two peaks coming so what is the difference between these two peaks is 120 ppm. Now this 120 ppm suppose we are doing an experiment at say 600 megahertz. So that means 150 megahertz is the resonance frequency for carbon now this we are talking about 120 ppm so in hertz how much it will be right 120 multiplied with 150 that will be something like this right. So this is the difference between this. So suppose by spinning if we introduce suppose we introduce a rotational speed which is matching with this or half of this then this will recouple recouple the interaction between C o and C alpha this is called rotary resonance condition now there are various other resonance conditions that we are going to talk and some of those is called redar recoupling. So this is the simplest one that I talked to just by introducing the spinning speed we can recouple some of the interactions. Now there is another recoupling scheme that is called redar this is recoupling for heteronuclear dipolar coupling by rotational echo double resonance. So this is a simple scheme that is done so we excite with 90 degree pulse on proton and then we refocus using 180 degree pulse here and for the recoupling what we do we select the tau rotor period and we keep applying this 180 degree pulse so 180 degree pulse series of that and then we have refocusing using 180 degree pulse in between and then we have a series of pi pulse. So this is called two phase alternating 180 degree rf pulse for every rotor period rotor period how you can calculate so we know the spinning speed so for one rotor period depending upon what is the spinning speed we can calculate the one rotor period and there has to be two phase alternating 180 degree rf pulse for every rotor period on the carbon on a heteronuclear channel and that basically averaged out the magic angle spinning phenomena that is there on the heteronuclear dipolar coupling. So by and then we apply 180 degree pulse that we had discussed about this in the middle of recoupling block on i channel that refocus the chemical shift Hamiltonian. So by doing this trick of reader we can reintroduce those interactions by recoupling sequence and there are various recoupling sequence that has been developed that have been developed that will be matter of extensive course on recoupling I will not go in detail of that I will just stick to basics of this. So some of the easy one of the recoupling sequence are called pdsd or dar or rfdr I will little bit discuss about these. Essentially what we do in this recoupling sequence we excite the proton using 90 degree pulse then we transfer the magnetization using cross polarization so now my magnetization is on carbon. Now suppose I am doing only 1d so this T1 at the moment just you forget I will introduce in a minute and then we do something called mixing. So we can mix it by proton driven spin diffusion that is called pdsd or dipolar assisted rotary resonance condition. So we apply a pulse here on proton which will be half of the rotors like rotational speed or about the rotational speed that will enhance the decoupling that is done in dar or rfdr radio frequency driven recoupling sequence. So we do that so rfdr is again series of pulses radio frequency driven recoupling sequence that basically reintroduce this some of the lost interactions that were introduced by MAS and decoupling and finally you acquire on carbon. So that is the recoupling sequence that is used. So now we learn how to decouple and how to recouple so one of the prominent proton driven spin diffusion basically this is something called second order recoupling for polarization transfer what we do basically you know we have abundant of protein in the protein. So here one amino acid here is another amino acid we have C alpha C beta C gamma C alpha C beta C gamma and carbonyl here. So at the moment we are doing only say these two nuclei we are in one day we are detecting on carbon while using the proton magnetization for enhancement of sensitivity. So can we transfer the polarization from proton to carbon and then detect on carbon with enhanced sensitivity. So by doing this what we are doing we are enhancing the sensitivity and also introducing the recoupling so that we have enough magnetization. So basically here spectral spin diffusion works on a phenomena called exchange of longitudinal magnetization. So what we do here we pulverize the magnetization from proton to carbon and then we decouple proton and then let them mix the late spin mix that we have seen in typical nosy spectrum. We put it in the longitudinal magnetization and let them let the magnetization mix. So that is what here happened but here actually the happen the magnetization is mixing through dipolar coupling and this is by something called flip flop mechanism that happens. So this using this we can utilize the magnetization transfer in homonuclear correlation experiment. So here we are detecting decoupling protons and letting them mix. So here is the mixing happening and then while decoupling proton we detect on carbon. So this is called PDSD, proton driven spin diffusion. By method of spin diffusion magnetization is getting transferred and we are detecting on carbon. Now this can come into 2D version as well. So here if we introduce a T1 evolution time that we have seen liquid state that will be converted into T2. So just to remind you what how we do 2D. It is a simple experiment more than one pulse we need 2 pulse. So here in 2D experiment you do series of 1D experiment. In 1D experiment what we have done? So we started with a proton and then we were detecting it. So this was 2 pulse experiment and in the second experiment we are increasing this distance between these 2 pulse. In third experiment we were further increasing this distance. So similar thing we are doing here into solid state as well. We are starting with a preparation state and then there is a mixing state and then we are detecting it. In the second experiment we are increasing the distance between this preparation state and mixing state. In third state even more, fourth state even more, fifth state again. So we are increasing it. So by increasing this time distance we are creating another time domain where a spin is evolving and then we are detecting in the direct dimension. So this is called indirect dimension, this is called direct dimension. So if you write it your omega 2 which we are detecting with a time this changes. You can see the peaks is changing and now we can do Fourier transform of these 2. So the 2 direct dimension and omega 1 indirect dimension shows a correlation peaks that we have seen. So this kind of concept is again employed in solid state for doing the 2D and that is what I showed you in previously. So start from proton, start with magnetization cross polarized on carbon and then we are introducing this time evolution. During that time we are decoupling it and then here we are allowing the spins to mix either by themselves which is called pDSD or by application of RF pulse which is called DAR. So by doing these we are introducing recoupling while the spins are mixing and then we finally detect while decoupling protons. So that is what is the 2D. Now we can establish the carbon-carbon correlation spectrum using pDSD or DAR experiment and that is what happens. So let us sum up what we have learnt till now. In solid state we have to take a rotor that rotor should containing all the biological sample whether it is in materials or amyloid fibres or membrane protein, we pack them in the rotors, we put the cap tightly and then we put in the magnet and in the magnet it is oriented at a magic angle at 54.7 degree and then we start spinning at desired spinning state with mass and then we are applying decoupling sequences we are getting sharp lines for each spins. Now we can extend this 1D information into 2D where establish the correlations. So this is now 2D correlation but here you see diagonal is only appearing so this is self-correlating. When we introduce this recoupling using RF sequence or pDSD so decoupling increasing the sensitivity and resolution, recoupling introducing again the interactions between these spins. So you can selectively introduce it now you can see these two spins are correlating so this is the 2D with correlation matrix. So that is what we are achieving spinning speed and cross polarization improves the sorry removes the anisotropy and removes the strong coupling it increase the spectral resolution and finally using recoupling sequence we are extracting the spectral informations. So using these things now we have transition from one dimensional solid state NMR to two dimensional which is the essential for getting any structural information. So let us see how our spectrum looks like 2D correlation spectrum. So like here pDSD or DAHR is carbon-carbon correlation spectrum you see it looks like exactly like a nosy spectrum that we have seen in solid state NMR and this looks like HSQC spectrum so this is NC correlation. So let me just explain you briefly what pDSD we were or NC we are discussing. So here we have N and C alpha right so this is the correlation we are. So here we have started with a proton transferred to carbon and then we are using the again CP condition we are transferring to carbon we are detecting sorry detecting on carbon while decoupling protons the one dimension the indirect dimension is N evolution and then indirect dimension is a carbon evolution. So that is what we are having here the carbon and nitrogen correlation. Now each of these peaks so if you are doing NC alpha each of these peaks giving idea about one amino acid. So each amino acid will have one NC alpha correlation so this is exactly like HSQC spectrum that we have seen in the liquid state that is the NC. Now carbon-carbon spectrum pDSD so what we are doing we are first polarizing all protons then using CP we are transferring to carbons these protons can transfer the magnetization to neighboring carbon or directly attached carbon and then we are mixing them so carbon mixing is happening and therefore you see lots of peaks are coming like a nose depending upon distance. So if you say take any isolated peak here are a trionine peak these are serine peaks so you can see C alpha C beta peak is getting correlated here C alpha C gamma peaks are correlated here serine has only two carbons C alpha C beta so that is coming here these all are actually the gamma and delta of isoleucines that are there. So the pDSD depending upon how long mixing time we are keeping how much dark power we are applying we can achieve longer correlation so all the way from C alpha to C beta to C gamma to C delta even the neighboring one like some of these will be neighboring interactions and these interactions like no G spectrum is very useful for getting the resonance assignment also getting the structural constraints. So just some more spectrum so we can even here we can have a HSQC kind of a spectrum. So in this experiment what we have done here that we have started with a proton transfer to nitrogen so like HSQC we had earlier right they started from proton transfer to nitrogen and then in indirect dimension we are evolving proton while detecting on nitrogen. So here we are getting H and 15 spectrum but things to be noticed here very importantly protons still has a broader line with you look at the line with this looks really elongated and if we compare this with NC spectrum this quite well resolved. So proton at the moderate speed still has lots of inherent problem of spectral overlap because the dipolar couplings are not averaged out but still we are getting quite distant spectrum in the solid. However if you look at the line width is quite huge about 250 hertz. Similarly we can do carbon 13 and 15 spectrum like all the carbon 13 HSQC we have. Similarly here we can do HSQC but again the lines are going to be broad. So here if you look at this is C-alpha, C-alpha and H-alpha correlation you can get C-alpha, H-alpha, C-beta all these methyl up to carbon proton correlation but line width are really broad. So we are not getting enough resolution here and it will be difficult to assign few of the peaks you can see here are still sharp these are coming from C-alpha, H-alpha and these can be easily identified. So here even though proton is in indirect dimension because the dipolar coupling could not be averaged out we are really getting broad spectrum and therefore the faster and faster speeds are required and lots of technological development happening in this direction. But at a moderate speed actually heteronuclear detections and heteronuclear correlations are of quite benefit. However we are almost getting liquid like spectral resolution using PDSD and using NCA experiment we can essentially get the resonance assignment. So people have developed various sequential resonance assignment like whatever we have in liquid just protons is removed in the name so you can have HNCO like it starts from proton transfer to nitrogen and then to C-alpha CO like these are the various transfer schemes NCOCA this is corresponding to HNCOCA this is H like H is not involved here CANCO, NCA-C-b, NCO-C-b, CANCO-C-b so you can transfer the magnetization from one nuclei to another nuclei to establish various correlation between N15, C-alpha, C-beta, CO and that is what helps us in doing the resonance assignments in solid. So what I am going to do in the next class I will be taking some of those methods how to go for resonance assignment but to give you an impression that if we assign all those peaks we can easily get the secondary structural chemistry like in liquid we have seen that depending upon what is the resonance frequency for C-alpha and C-beta we can find it out whether they are upfield shifted or downfield shifted like suppose we have a beta sheet in beta sheet the from the random coil the C-alpha will be upfield shifted and in alpha helix it will be downfield shifted C-beta will be reversed so we can assign those and then subtract the random coil from those value plot along the sequence and we can find it out the secondary structural information of the proteins polypeptide chain. So you see by doing these experiments we are all the way arriving at the secondary structural information using solid state NMR techniques. So here I will stop to by giving you impression that solid state NMR can be used for a structure determination. I will go little more detail show you what are the pulse sequence in details how you can use those strategy for resonance assignment in solid and how you can get the structural parameters for getting the structural information as well as the dynamic information in next classes. Thank you very much.