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Module 1: Protein Sequencing

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Now, all these techniques are the Sanger method, be it the Edman’s degradation method or be it the pure mass spectrometry. All these methods usually work better or are usually applicable only for short peptides. If you use a very long protein sequence that has 500 amino acids for example, very long protein sequences that cannot be used as a sample for all these methods. So, that is the limitation of the protein sequencing methods. (Refer Slide Time: 10:18) So, large protein sequences cannot be directly used as a sample for protein or peptide sequencing methods. So, that is the limitation of these techniques, large proteins or original proteins that you have isolated from some source cannot be used directly to determine it is the amino acid sequence, what these methods are usually for Sanger’s method, Edman’s degradation method or the mass spectrometers method. They usually use shorter peptides sequences. So, sequencing methods, peptide sequencing methods right now, peptide sequencing methods are useful for shorter peptides such as maybe 40 to 60 70. Let us 60 amino acids long, so 300, 400, 500 amino acids long protein sequences cannot be determined short sequences of peptides are the best as a sample in these methods that you can use these short sequences to find out they are exact amino acid chain length. So, what is the way out? How can you then, if you cannot use the full protein as directly that are using these methods if you are not able to determine the sequence of the full protein, then what is the way out to understand the sequence of this full protein. The idea is what you have to do now is if you have a protein, full-length protein that you have to break down into short pieces of the peptides, which will have in this length maybe 40 to 60 amino acids length. So, protein first degraded into shorter lengths of peptides. So you take your protein sample treated in some way so that that produces a number of short peptides. Each of them may have the or each of them would have the length below 40 to 60 amino acids and then all those fragments, all those short lengths of peptides that you have made out of this protein can be used separately to find out the sequence. After this, you can use the advanced degradation method to determine the sequence of these shorter fragments. (Refer Slide Time: 14:47) So, in this case, you have to break down first the large proteins to shorter peptides, which is very important. Now the question is how to do that. So I will show you 2 methods. There are actually 2 ways of doing that. One is the enzyme meeting method. Second is the chemical method. So first let us take so there is the enzymatic method, and the second is chemical way to break down a protein into pieces. The enzymatic method, so you have to use an enzyme that can cleave a protein into pieces. Not only that, but it also has to be specific the enzyme has to be such that it will cleave the protein at specific amino acids, then only you can know where the cleavage is occurring it cannot be arbitrary cleavage, it has to be pre-determined cleavage. So, that you know where my bonds have been broken and then you can take the actions accordingly. So, such kinds of enzymes that cleave the proteins are known as proteolytic enzymes or they are also known as a peptidase. Peptidase means that it cleaves a peptide bond. There are many examples of such peptides or such proteolytic enzymes that are present in our body, that are present in other animals that are present in plants. And they have a very important application actually, especially the proteolytic enzymes, or the peptides are very important in our metabolic system. When we eat some food, protein-rich food for example, then because the protein is so long, it is difficult to get digested. So, these peptidases of these proteolytic enzymes would cleave your food or cleave the proteins present in the food into short sequences into pieces, and then your digestion or the metabolism becomes easier. So these kinds of enzymes are present everywhere. I will give you some examples. So papain, piping or papain is one such enzyme that is present in papaya. And this enzyme cleaves the proteins into short sequences. So, I do not know it says school chemistry. If you, have noticed that when you cook food, some especially the protein-rich food, for example, meat. If you add papaya to it, then it takes less time to boil or it takes less time to cook the food. Your meat gets soften very easily if you add papaya there. So the reason is this because you get the enzyme papain, which is a proteolytic enzyme and that cleaves the protein in the meat into the smaller fragments. And then your cooking becomes easier. So other kinds of examples of the proteolytic enzymes are, which we will be using now is trypsin or chymotrypsin. So, this is one proteolytic enzyme, this is another proteolytic enzyme. Trypsin and chymotrypsin are usually that you find in the market are usually isolated from pigs. So, how are they specific to sequences where do the cleave in the protein structure. (Refer Slide Time: 19:44) If you use trypsin it actually cleaves the peptide bond because it is the peptides, so it cleaves the peptide bond at the C terminal position, C terminal of the amino acid either lysine or arginine. So if you have I am starting somewhere in the middle, this is the N-terminal NH. This is your R 1 R 2 if you have a lysine here or an arginine here income CO NH R 1 R 2, so, this is R 3. So, I will write R 4 here and so on it goes on. So, this is the C terminal. This is the N-terminal. If you treat these with trypsin then it cleaves at the C terminal of lysine or arginine. That means, here this peptide bond, this is the peptide bond after lysine, and this is the carbonyl group. So, this is the C terminal part of the lysine. So, after lysine, the following peptide bond would be selectively cleaved by the enzyme trypsin. It can be either lysine or it can be arginine or both. So, wherever there is lysine, wherever there is arginine the following peptide bond would be chopped off, by trypsin. So, what you will get, you will find amine R 1 CO NH R 2 CO NH lysine this is cleaved. So, you have a free carboxylic acid here plus your protein is now fragmented into 2 pieces, at least one is this, this here. Another one is this amine R 4 CO and the other part, the rest of it. So this would be one fragment, this would be another fragment. So that is how trypsin would cleave at a specific amino acid sequence in the protein. So, if you take a whole protein and if you treat it with trypsin, then you can know where your trypsin is cleaving, which amino acids or which peptide bonds this is cleaving. And the fragments that you get, after the reaction that you can isolate each one of them you can separate out. And each of them, you can put it or you can use further for Edman's degradation method. (Refer Slide Time: 23:57) So, once you have the fragments, the next step is you purify each fragment and then use it for Edman's degradation. So, in this way, you will know the sequence, exact sequence of each fragment and then you can basically tie them up to get the full sequence of the protein. So, that is the mechanism of action of trypsin. Now, if you have chymotrypsin, chymotrypsin is another proteolytic enzyme or another peptide is and it cleaves the peptide bond at the C terminal end of tryptophan Trp. So, if you have a protein sequence, if you treat it with chymotrypsin wherever there is tryptophan, it will leave the following peptide bonds and you will get the shorter fragments peptides, again you can purify each of these fragments and then use them for Edman’s degradation method. So, that is how you know whenever you will be using an enzyme you have to select an enzyme for which you know where it cleaves. That is very important actually a predetermined sequence has to be cleaved. (Refer Slide Time: 26:11) Now comes a chemical method. In the chemical method, we use a reagent of course, and this reagent is CN Br cyanogen bromide. This is the region that is used to cleave a peptide bond at predetermined amino acid. So this against cleaves the peptide bond again at the C terminal end or C terminal position of specific amino acid. In this case, is methionine met? So the same here cleaves all the peptide bonds wherever there is a methionine. How does it work? If you have so this is N-terminal there can be other peptides, other sequences here this is R 1 CO NH. Let us say here you have a methionine CH 2 methionine is S and CH 3, this is methionine. This is the C terminal end. So this is the C terminal peptide sequence R 1 R 2, this can be R 3 and it goes like this. So, it should be CO and then the rest of it. Let us say that this is the sequence of the protein. And if you treat it with CNBr, then what happens, bromide is a good living group. It is the electronegative group. So it will try to drag the electron density and move away, sulfur has one pair of electrons, and sulfur has a large size. So it has the one pair of electrons is highly donatable because it is to some extent electropositive. So the one pair of electrons will attack the carbon, attack the carbon of the cyanide eliminating the bromine or the bromide. So, you will have CO NH here CH 2 S CH 3 and cyanide and this would be now plus because it has donated the electron So, sulfur plus, in each R 3 run this, now this has formed a polypeptide here and it has this, but if you look at cyanide that is why cyanide has been used cyanide or CN minus has a dual character. So, in this case in cyanide, cyanide also is pretty stable as cyanide minus. So, it has the tendency also to drag the electron density towards itself because nitrogen is electronegative. So, the electron density would be dragged towards the nitrogen that forces the electron density of this bond to be dragged towards the cyanide, that makes the sulfur, even more, electropositive, even more electron-deficient, it has already a plus charge and because of the cyanide attraction, electron-withdrawing effect, this the positive charge would be or positive charges density will be higher here. So, this whole thing becomes a very good leaving group. Since this is highly electron-deficient, it will try to take away the bonded electron and move away. So it is very easy to cleave this bond. So, if you hydrolyze it in presence of water, the carbonyl here, that is where the chemistry of the peptide bond is coming. This although it is not very reactive, a peptide bond is not very reactive, but because you have a pretty good living group, this reaction happens, it will leave, it will attack this carbon eliminating the whole thing, the sulfur. (Refer Slide Time: 31:54) And you get a cyclic compound which will look like this is CH 3 CN. This is a positive charge that makes it a good leaving group CO NH yeah this is R 1 R 2, this is R 3. So, that will attack this carbon and they should leave. So, what you have is of course, this is with treatment with water R 1 CO NH, this O here. So this bonded electron go and then this would be the resonance, this is gone, this is coming here, this is attacking. This is the carbon and this would be your NH. This is your R 3 CO and the rest of it. Here you have formed the double bond and would be NH plus. So, this is the cyclist compound that will be forming through the oxygen, and to stabilize this, there will be this resonating structure. Now, this becomes a double bond with the positive charge on NA amine or with the positive charge on nitrogen which is also not very stable. So, it will undergo rapid hydrolysis and this bond would be cleaved. If you hydrolyze a carbonnitrogen bond what you will have water only attack here will move this. So, you will basically get a carbonyl compound here CO NH, this here will have a carbonyl plus you will have the free amine H 2 R 3. So, you have breaking or the cleavage of the peptide bond in the position where there was a methionine or the following peptide bond after the methionine. So, this is the chemistry here. And now you can again separate the individual fragments and do the Edman’s degradation. So, the number of fragments will tell you. So if you have 5 fragments, for example, in the whole protein sequence, then you know, you have at least 5 methionine, present in this at the terminal of the, wherever there was the cleavage, okay. So that is how you can actually break a given protein into its shorter peptide sequences. (Refer Slide Time: 35:43) So, now I will give you 2 problems. Problem 1is let us consider a protein sequence N-terminal glycine, aspartic acid, phenylalanine, lysine, tryptophan, arginine, phenylalanine, alanine, methionine and this is your C terminal. Let us say this is a given protein sequence. Question a, is write the fragments if treated with trypsin. Question b; write fragments if treated chymotrypsin. So, can you write down the fragment of the proteins, or what are the fragments that we will obtain if you treat this given sequence which is trypsin? What will happen if you treat it with trypsin, it will cleave the peptide bond at the C terminal position of wherever there is a lysine or wherever there is an arginine. So glycine, aspartic acid phenylalanine, lysine, tryptophan, arginine, phenylalanine, alanine, methionine. So, if we treat this with trypsin then it will leave here. There is a lysine here. So after the lysine, you have another arginine here to cleave keep also here. So, the fragments that will see are lysine, aspartic acid, phenylalanine, lysine, free acid plus N H 2 tryptophan, argentine free acid plus it will expose the amine at the phenylalanine methionine. So, this is the C terminal end. So, this is the C terminal. So, these are the 3 fragments that you can expect if you treat in this specific protein sequence with trypsin. (Refer Slide Time: 39:34) Now b is glycine, aspartic acid, phenylalanine, lysine, tryptophan, arginine, phenylalanine, alanine, methionine and you are treating this with chymotrypsin, and we know that chymotrypsin cleaves the tryptophan, the C terminal end of the tryptophan, so it will basically cleave here, cleaves after tryptophan and there is only I think 1 tryptophan. So you will have a single cleavage here. And your fragments would be glycine, aspartic acid, phenylalanine, lysine, tryptophan. This should be chopped off it will expose free carboxylic acid plus free amine of the next arginine and the rest of it, alanine, methionine. So these are the 2 fragments that you can expect from here. (Refer Slide Time: 41:23) So, I will give you another question. Let us take a different sequence alanine, glutamic acid, arginine, valine, leucine, methionine, phenylalanine, Trp tryptophan, alanine. So, this is the given sequence. Question a is if you treat this treat with CNBr then what are the fragments CNBr followed by, of course, this is very important followed by hydrolysis, treatment of this sequence with CNBr followed by hydrolysis, what are the fragments that you will find. CNBr will break after methionine right. So here this should be broken and you will end off with alanine, glutamic acid, arginine valine, Leucine, methionine, of course, is no free carboxylic acid, it will finish here with the cyclization. So I am writing this as cyclic. You do not have to write anything actually. So it is methionine plus it will expose the amine of the next amino acid which is phenylalanine, tryptophan alanine. And that is your C terminal. So these are the 2 fragments that you will obtain. The second question is first to treat with trypsin and then with CNBr, so it is a combination of treatment with the enzyme as well as the chemical reagent, you are treating both with trypsin and with CNBr, then what are the fragments that you can expect. This of course with the expectation that they will not interfere with each other business. So, if you treat it with trypsin what you will have, you will have a cleavage of arginine, you will have the only arginine here. So, let us write that alanine, glutamine, arginine COOH plus NH 2 Valine, Leucine, then you have methionine, you have used CNBr. So, it will cleave after methionine to stop there plus amine, phenylalanine, tryptophan, alanine. This would be your products. These are the 3 fragments that you will obtain. I guess I was correct trypsin, arginine. There is the only arginine no lysine, so only 1 cleave is here for this and another for methionine 2% 2 cleavages that will give you 3 components. So 3 fragments that you can expect by a combinatorial treatment of trypsin and cyanobromide or cyanogen bromide, this plus, this plus this. So, likewise, you can actually find out you can play around with the different sequences. So, if you take a large protein sequence and treat it with the combination of trypsin and cyanogen bromide or even a combination of trypsin plus chymotrypsin plus cyanobromide, then what are the fragments. You can expect the huge number of fragments that will be present in your final products and you can separate each one of them, you can expect that your protein is now degraded enough into the short sequences, which you can use for Edman’s degradation method to know its sequences. So, this is roughly about the sequencing of the proteins, if you have an unknown protein sequence, how would you find out it is the amino acid chain. What are the amino acids present one after the other? We have talked about 3 methods for the sequencing itself. And then today, mostly we have talked about the methods through which you can actually cleave or you can actually make fragments from a large protein into its smaller pieces.