 So, that is why we need a system which can give me an idea how the structure behaves in its solution structure. And for that, we have CD spectroscopy, which can give me a unique idea how many alpha helix I have, how many beta sheets I have from a solution. How to do that? So, as we just said, before I do a CD, I need to do absorb a spectroscopy first. And over there, say I have a peak like this, and this peak generally look into 180 to 230 nanometer region, every protein have a band over there, every protein, because this band is coming from the amide bonds. And as you know, amide bonds are actually present in every protein. This band will be always there. But with respect to that, if I now take a look into a CD spectra, how the CD spectra looks like. And here comes the data, how it actually looks like, that is how it does look like. So, let me go through one at a time. So first, I look into the ellipticity, positive, negative. Don't look into the actual value now, look into the trend with respect to the lambda. But if you have a alpha helix, if you have a alpha helix, you are going to have a signal like this, a very characteristic signal, same as the blue line over here. So, you have a positive band around 193 nanometer, then a negative band, it crosses and comes to negative. Negative region, you have two maximas, one at 208 nanometer, one at 222 nanometer. If you have these three signatures, 193 positive, 208 and 222 double negative, you can say, I have an alpha helix. For beta sheet, how it is coming? So, for beta sheet, you have a positive band around 195 nanometer. Then it goes to negative. However, instead of double negative, you have a very broad peak and the maxima comes around 280 nanometer. And if you have that, you can say, I have a beta sheet, one positive 195 and one broad negative at 280 nanometer. And then, if you have a random coil, that means not really oriented in any particular way, it starts with a negative value around 100 lower value and then it crosses and then it remains positive in the region between 210 to 230 previously, which was in the negative region for both alpha helix and beta sheet, now it becomes positive. And there is no particular such peak, it is mostly slowly going down. The peak only founds over here 195 nanometer, but it is now in the negative region. If you have that, you can say I have a random coil. That means if you take a sample of only alpha helix, you should see a band 193 positive, 208, 222 negative, you say I have alpha helix, beta sheet, characteristic peaks, 195 positive, 218 in negative, random coil like this. And it is going to give you the data in the solution. Now you can imagine that in a protein, there is a mixture of alpha helix, beta sheet and random coil, right? So what you are going to get, it is a mixed signal, you are going to get a mixed signal in CD, this is absorbance spectra, this is CD spectra, I am also looking at the CD spectra now and what you are actually going to get is probably something like this, which shows that you have a mixture of alpha helix and beta sheet. And you have the particular values and from there what you need to do, you just need to do some deconvolution, where you actually try to get the original data and try to match up with some expected values. Say I have 70% of my alpha helix and say 30% of that is beta sheet, if they matches then I can get this black color original data. So from there I can find out how much is my beta sheet and how much is my alpha helix, you can find out and also you can find out how much is your random coil. And all those things you can imagine now you can be done computationally, you have to just record the original data and fit the data into a computer and ask them to run a logarithm to find out how much is alpha helix, how much is beta sheet. And by that you can figure it out what is the overall structure of a protein in a solution. And not only that you can also figure it out what is the change happening in that particular molecule as you change the pH of the solution, as you change the temperature of the solution and that will be directly affect the alpha helix, beta sheet, their orientation and the CD spectra will change accordingly. So for an example say I have a protein structure with only alpha helix and I have a data like this. So in anywhere in your life you are drawing a graph make sure that you always draw the accesses and write the proper accesses like what are those accesses actually mean. So in that I see this particular data at say 298 Kelvin. Then say I change the temperature I am heating it and slowly the same solution data looks like this. Then at one point of time it becomes like this. That means what is actually happening as I increase the temperature my alpha helix structure is going to a random coil. So what does it says? It says my protein structure is actually degrading and if I can measure this particular data at different temperature and say I am monitoring what is the change happening at 208 nanometer and plot that with respect to temperature how my data will look like at 28 nanometer I have a very negative value at 298 Kelvin then I go for the temperature high and slowly and slowly it is going up and at one point time it will remain same because at that point of time you actually already degraded the protein. So from there this point I will say here the protein is breaking down and this is known as the melting temperature of a protein. melting temperature means where a particularly nice helical structure with respect to temperature just defaulted and protein folding has a huge role to play with its function. So with respect to CD data we can very easily find out exactly what does it happen and if I want to do an experiment at which particular condition I should do where the protein structure is still stable and after what I can actually break it down. So what is the temperature I should keep my protein so all this fun example right now we are looking into different vaccines and some of the vaccines are protein based vaccines and over there you can imagine that they always have to keep it at a particular lower temperature why because if you go to higher temperature you can break down your protein structure and the vaccine will go bad. So how to look into that how to find it out which temperature will be the safest for it so you just do a simple CD spectrum find out where you see this melting point and you can specifically point out this is the temperature you have to keep to make sure that protein is stabilized. Now the last part I am going to talk about yes sir you showed in graph that alpha helix is changing to that upper one graph in random coil but what about beta seed. Yeah I am just giving an example it can go to a beta seed it can all the possibilities can be there because I am just giving an example that one alpha helix is going to random coil you can think about it can also happen some time higher temperature can actually bring you to totally new structure it can go to alpha helix to a random coil to a beta seed and from the CD spectrum you can follow it up like exactly what is actually happening. Means after hitting beta seed can be converted into random coil yes it is possible all possible. So random coil is generally the structure at higher temperature because it does not have any hydrogen bonding or anything over there because hydrogen bonding is actually stabilizing it right when you heat it you are giving it enough energy so that you can go and break those bonds because entropically over here you are losing some energy but enthalpically you are getting some energy so that is why the delta G is negative to form a such structure structure in alpha helix or beta seed but in random coil you have a huge entropy so that is the favorable thing but there is no hydrogen bond over anything to enthalpically help it delta H is pretty actually non-existence over here however at high temperature you are giving enough energy from the outside so only entropy can take care of the delta G and you have to have enough delta G enough energy coming from outside so that you can break these bonds and over their delta H so from there you can even find out what is the hydrogen bonding energy present in the alpha helix or beta seed and in some proteins it might happen you have five alpha helix two beta seed when you heat it only two of them breaks down not all of them and from the city spectra you can find it out exactly which of them are actually breaking down. So there are all different kinds of possibilities out there the main point is that from the city spectra you can follow it like what is the change happening by following the alpha helix beta sheet and random coil signatures okay because this particular data I just showed over there they are signature no matter what where you measure a alpha helix always will show this particular beta sheet will always show this particular peaks okay any more question sir I didn't get about the peak that is near 125 positive peak. He said 125. Sorry 195. So what do you mean so what is the question 195 so that is actually positive signal so that means over here this is AL minus AR right a function of that that means over here AL minus AR is a positive number so that means your absorbance of the left hand circular polyesterite is more than the right hand circular polyesterite and then you when come to 208 222 around this region AL is actually absorbed less compared to the right hand circular polyesterite so that is it becomes negative so that is what we mean by positive and negative that is the direction of the value of the shy thank you sir okay any more question yes sir from that value how can we decide that that is due that is for the alpha helix or beta sheet means how we confirm that means that value but I want to know that how is the characteristics is decided from that value that means that for the alpha helix it is decided that 193 or we'll get that but I will confirm that that is due to the alpha helix only or when the first time is done how will get that the alpha yeah so mainly yeah mainly people actually look into this negative region to be honest because this 208 222 there are very unique two humps beta sheet have a very broad peak and random coil is in the positive side so people mostly look into this region 200 to 230 region and over there then you have a result like this and then you try to deconvaluate it put different values for alpha helix and beta sheet and see which data by calculation is matching the original data and from there you can calculate like what is the probable alpha helix and beta sheet distribution for that particular protein does it answer your question or no point yes for example if you take 70 percent alpha helix and 30 percent beta sheet versus a 50 percent alpha helix 50 percent beta sheet the overall data will not look same because these three different humps because there are three different humps when both alpha and beta helix are present 208 218 222 and their contributions will vary with respect to the overall presence of the alpha helix and beta sheet and that is what we actually follow that what is the distribution of these three different humps at 208 222 and try to match it how it matches with the actual experiment and from there we find out how it actually happens