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Amino acids and Proteins

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Hello everybody and welcome back to the biomolecules. So, we have been discussing the structure and properties of amino acids, peptides and proteins. Most of the biological reactions are we know are catalyzed by enzymes.And as you know that there are around 21 naturally occurring amino acids in nature and these are the structures. Out of those 21amino acids, one amino acid that is selenocysteine is not a coded amino acid which means only 20 amino acids are coded by genetic code. There is no genetic code present for selenocysteine and it is a non coded amino acid. But Solanocysteine is found in many proteins and many enzymes. But it is not necessarily present in all organisms because all organisms do not have Solanocysteine in their protein structures. So, it is not a very common thing if you look at the structure of the amino acids it has a lot of variety and a lot of chemical variations in their structures. For example, you start with glycine has one hydrogen here so it is a CH2 here for all other cases and this CH2 is CH is replaced by some substitutions and all of this amount naturally occurring amino acids have L configurations except of course the glycine. Glycine is a chiral all the other amino acids are chiral amino acids this amino acid is not chiral or a chiral because it does not have a chiral centre all other amino acids have a chiral centre. And all of them have L configurations; basically, all naturally occurring amino acids have L configurations. And we have mentioned prior that the nomenclature, of course, came from the glyceraldehydes. If you look this is the structure of glyceraldehyde and this has L configurations sorry there is an OH group. An OH group is on the left-hand side so that is why it is called the levorotatory or has L configurations. Similarly, if you look at all the structures of the amino acids they have an equivalent structure of the glyceraldehydes, in this case, this is C double OH. here I am writing R for general and H for amine. And NH2 is on the left-hand side so they all have L configurations which ideally means the S configuration. The chemical nature of the amino acids are very different from each other, there are a lot of variations as I was saying. So in this part you can see that this is their alkyl substitutions with branched chains there is a cyclic system in proline which has a second or a minor acid and that makes it very rigid. There are functional groups their hydroxyl groups here which can be functionalized ethyl group here there is a sulphide linkage amide bond here, here. And there are aromatic rings substitutions with the heterocyclic rings as well. And there are the acids and the basic functional side chains like lysine and arginine have a mean side chains lysine has a mean side chain arginine has a guanidine as a side chain. They are basic side chains; aspartic acid and glutamic acid have the acid carboxylic acid groups as the side chains. So, there are a lot of variations which allows you to chemically modify the amino acids enormously. The basic structure of the amino acid if you write it in the 3-dimensional form you can write it in any other way also. I am writing this way where you have the up the plane H down the plane and this is the carboxylic acid end and that is the I mean so this is the general structure of amino acid. And of course, the reason it is being called amino acid is that it has an amine group and a carboxylic acid group. So as a group, in amine group amine is base and in the carboxylic acid group, it is an acid group and that makes it have our different structures that I am coming very shortly. If you write a peptide out of it then you will and see not writing the all of it CO and in H then this will come down the plane R1 R R1 CO NH this will go up again R2 and it will go the end is a carboxylic acid and here can be the amine so it goes and the peptide is drawn in that fashion. You can draw in many other ways and you can see that they have a peptide bond which is an amide bond. Now, this peptide bond exists in two different structures it can have resonating structures. So, if you look at the peptide structures you see that this bond has a partial double bond character. And apart from this bond other won't like this and if you see this bond and this bond here this bond and this bond here they have pretty much a single bond character. So ideally if you look the backbone of the peptide or backbone of the protein structures there are plenty of single bonds. A protein has a very flexible structure because free rotation around a single bond is allowed. And you can ideally rotate the all the single bonds to any directions and to any angles. So, you should have a very flexible moulded structure of a protein. But in reality the structure of protein is not as flexible as you think, lot of restrictions are there in the orientations. There are two basic reasons for it number one is, if you look here at the peptide structure then you will notice all the single bonds that you look here are not the single one and there is this peptide bond which has the conjugated character as the nitrogen lone pair that makes resonating structures gives you the double bond character. Reason number two is there are two planes if you look at the structure here. For example, if you will see this bond this is the amide bond which has the double bond character this is a single bond. So, this makes one plane which is given here, on the other hand, this one makes another plane. And if you look at this plane there is a certain angle between these two planes and this is known as the dihedral angle. The dihedral angle between the two planes, so, if you change the plane ideally you should be able to change the angle because the free rotation around the single bond is possible this is the single bond and they are named as this is called Phi and this is called Psi. So, you should be able to rotate Phi and Psi too freely. But we will see later on just in a while that if you rotate those molecules then the atoms they will sometimes bump into each other and that makes the free rotation around these single bonds not allowed or restricted. So all rotations around the single bond are not allowed and that was given by a plot called Ramachandran plot, that I will just discuss it in a brief while. So, our second thing is that we have seen for DNA that there is a way to write the DNA structure. So, we have seen that you start with the 5 prime ends and complete it at the 3 prime ends. Similarly for peptide, there is a way to write. NH 2 CO NH R3 OH, so you will see that as you move on the right-hand directions you can continue here when you complete the chain there will be a free carboxylic acid on there on the right-hand side if you write in this direction. On the other hand and there will be one free amine group on the left-hand side and this is the way to write a protein structure or a peptide structure. That usually from amine terminal and ends up with carboxylic terminal. And this is called the N terminal in a terminal of the protein and this is known as the C terminal. N terminal for amine terminal C terminal for carboxylic acid terminal and this is usually the practice that we use to write or to represent a structure of a protein. So, before going to the whole protein structures I will come back again to the amino acids and there is a property of amino acids which you already know that I will just briefly discuss. So, I am not writing the stereochemistry now R C O O H I mean this is the structure of amino acid. And as I have mentioned that it has a basic group which is the amine functional group and it has the acid group which is the carboxylic acid group. So, when you keep a base as well as an acid together mix them then what do you get for example if you mix HCL and sodium hydroxide then immediately you will get a salt sodium chloride and water. So, HCl if you take that is a strong acid and sodium hydroxide that is a strong base if you mix them you will get a sodium chloride as the salt plus water. So acid and base together cannot coexist they will form a salt immediately. For amino acids what will happen there are organic acids and organic bases so there will be proton transfer from the acid to the base. So, this molecule does not exist in this fashion, it mostly exists in this way NH 3 plus and COO minus. This is what called the zwitterionic form or zwitterion. If you look at the net charge, of course, the net charge is 0 but it exists in the ionic form. So, this one does not exist much. The free form of the amino acid exists in very very less quantity, most of the concentration of the amino acids will be here in the zwitterionic form. And there is one that is where a property called the isoelectric point is coming, it is a characteristic of an amino acid that is known as isoelectric point usually represented by PI. So, isoelectric point is a kind of characteristics of an amino acid that is defined as if you have PI is defined as the pH at which the concentration of the zwitterionic form is maximum. So, as you vary the pH of the solution then, of course, the acid group is there the basic group is there and depending on their acidity and basicity the structure should be changed and the proton transfer phenomena would be changed. So this is the pH at which the zwitterionic form that is the net charge 0 is the maximum will have the maximum concentrations. Now the question is how to calculate the isoelectric point and it varies actually from amino acid to amino acid. So, if you take for example the structure of alanine, alanine has a CH3 here say carboxylic acid. And so how do you calculate the isoelectric point of an amino acid? I am just taking the example of an alanine. So, usually, it is done by a titration, you do a titration of the amino acid against a strong base sodium hydroxide. Usually sodium hydroxide, potassium hydroxide can also be used so strong base against the amino acid. Then and you start it with a very low pH and then see how it is going what are the changes that you are having. So, if I now draw the structures of the amino acids starting with the very low pH then what are the structures that are possible or what are the equilibriums that are possible and then how can you find out the PI from there. So, if you start with a very low pH, acidic pH then all of the functional groups' acids will be acid, of course, the base would be protonated because this is dipped in strong acid. So, the initially what we'll have you will have an NH3 plus and COOH in very low pH and then as you increase the pH as you tighter it against the in sodium hydroxide you are adding sodium hydroxide slowly then that is raising the pH then after a certain pH what will happen is an acid which has a low pka value. So, that will be deprotonated first you will get this negative charge here this is the acidic group not easily deprotonated. So, this is known as pka1 this is the first pka then as you move further on this is minus eventually this should be deprotonated also the NH3 plus and will become in NH2 so this is your pka minus the second pka so the first pka is for the carboxylic acid and the equilibrium is from the free acid to the carboxylate anion the second pka is for the deprotonation of the NH3 plus to NH2 the basic. Now if you look at the values of the pka, alanine so your value of PI would be the average of the two because the concentration of zwitterion around you are calculating the concentration of the zwitterion which is this net charge 0 plus charge minus charge net 0. And this is involved in both the equilibrium this step as well as this step. So, if you want to calculate the concentration of the deuteron you have to take the average of both the equilibrium. So ideally PI is calculated as pka1 + pka2 divided by 2 so if I take the example of glycine I, have the data for glycine here you can write the same thing the first one COO minus NH3 plus and then this equilibrium minus and this becomes NH2. Now, this pka value is around 2.3 and this pka is around 9.6. so, your PI would be calculated as 2.3 because this is acid so low pka and this is basic because pka showing above 7 and 9.6, in this case, 9.6 divided by 2 or it is coming as 5.95. This is the PI of glycine and it is pretty much our characteristics of glycine. Now things are a little bit different when you have other functional groups so you have acid and base so if you have a functional the sidechain as another acid or another base then how it would look like how the how would you would calculate the PI. So, if we take the example of aspartic acid that is aspartic acid is this there is a CH2 COOH carboxylic acid sidechain. This is acid of course so at very low pH everything would be protonated. So, this would be your first form at very low pH. Now as you increase the pH as you add more sodium hydroxide what will happen your acid would deprotonate but it will go stepwise because the pKa value of this acid and this acid are different. And in fact, the pKa of this is low for glycine case we have seen the pKa of this is around 2.3 and you know so this is a derivative of acetic acid kind of thing acetic acid has a pka value of around 4.7 so this would deprotonated first minus NH3 plus. Next is this carboxylate would be deprotonated this acid would be deprotonated minus NH3 plus and there would be another equilibrium where everything would be deprotonated. This so this is pka1, pka2 minus and this is pka3 now the question is which steps you will consider to calculate the PI ideally we are trying to calculate the concentration of the zwitterionic form. So, where is the zwitterionic form where the charge is 0 net charge is 0. So, here if you calculate the charges the net charge is here +1 this is where OH is 0 this has a charge of -1 this has our charge of -2. So, if you have to calculate the zwitterionic form concentrations you have to take this two equilibrium where they are involved pka1 and pka2. So your PI for aspartic acid would be I am writing an SP aspartic acid would be pka 1 + pka 2 divided by 2 just like the earliest and I have the number only this is one point value is 1088 this value is 3.65 and PK 3 this value is around 9.68. so your pk1 is 1.88 pk2 is this +3.65 so it is already different than glycine because glycine case and this one was coming into picture divided by 2. So, the average is around 2.76 this is the PI of aspartic acid, of course, it is way different than glycine has 5.9 in this case aspartic acid has 2.7 then that is because it has a carboxylic acid as the sidechain. If you have instead of the acidic side chain if you have a basic side chain then what will happen you have this I am drawing the structure of lysine. Lysine as 4 CH2 here and then an amine acid NH2 so again at very low pH everything would be protonated so this would stay as NH3 plus this should be NH3 plus as well so the first equilibrium would be deprotonation of the carboxylic acid 4 CH2 here NH3 plus this should be minus NH3 plus. The second one would be deprotonation of either of this amines. So, it turned out that the pka of this amine is less. So, this should be protonated this one would be deprotonated becomes NH2 and then the sidechain amine this would be deprotonated minus NH2. So, this is your pka1 which has the value of around 2.17 pk2 has the value of 9.04 this is pka2, pka 3 this is around 12.48. Now the question is what will be the PI how which steps you will consider for the calculation of the PI. Again you try to find out the zwitterionic form by calculating the charge. So, this has a net charge of plus 1 plus this is plus. So, this is +2 +2 here you have +1 2 positive or negative here you have one negative one positive this is your 0 charge and in this case, you have -1 so your change of the charge is from +1 to -1 via 0. So, if you want to it is a usual practice to find out the PI of amino acid. What you look for is where the charge is changed from a positive to negative via 0 or it can be another way around negative to positive via 0. So, that steps should be considered so your PI would be here is the digital unique form which is so this equilibrium and this equilibrium should be considered. So, it is pka 2 + pka 3 divided by 2 in this case 9.04 + 12.48 divided by 2 that is coming at 10.76. So, this is the PI value of lysine, of course, it is very again because it has a basic sidechain so the PI value is high. So, that is how you calculate the zwitterionic structures or the maximum concentration of the zwitterionic forms. So, now how do taste up I am I know acid how would you identify and when the presence or how will you taste the presence of an amino acid or a protein. So, this is a classic test that is usually been done to find out whether this is a protein or not this is called anine hydrin test and probably you have heard that name and you know this is the same test that is used for fingerprint detection in crime thriller or programs you have seen that the people are they are spraying something and then they are fine trying to find out the fingerprint or from there. They see certain colour usually this is a violet colour or the pink or the purple colour that appears when you spray that and they spray the Ninhydrin solution and that is to figure out whether somebody has a fingertip on it and a fingertip means our even our skin has proteins and amino acids so if you press it somewhere some of the proteins are very trace amount of the protein, would be labelled there. And it is a very sensitive test to find out whether the amino acids or proteins are present. So, it goes like this is the solution of Ninhydrin this is called Ninhydrin and here I have shown using the amino acid, not the protein. If you have a free amino acid then how the test goes what is the reaction that happens. So, first is if you have the ninhydrin here your OH it is a diogen and diol basically and then you have amine group this is R. So, the first reaction is the lone pair I mean is basic and it has a free lone pair of electrons it is nucleophilic. So, it is a nucleophilic substitution this goes attacks and one OH should be eliminated. And because the carbonyl groups are there they drag the electrons that make the carbon more electrophilic. So, the nucleophilic substitution of the amine becomes very facile OH you have now NH C O O H and here is your R. Now once again another one this goes and another way elements so it is minus two molecules of water basically and that gives you this structure a double bond and then you have a carboxylic acid so it gets decarboxylated if you have OH all right. Here this goes it comes back here because again since you have electron-withdrawing carbonyl groups they have the tendency to withdraw electrons that makes this electron deficit. So it can attract electrons and this carbon can attract electrons. So, you will get this form first with a double bond here and then it gets an originating structure this is more stable. So, it will form this compound. And now the presence of water will hydrolyze this bond it is very easy to hydrolyze this bond. And if you hydrolyze this you get ideally our aldehyde and an amine. So, R free amine would be created here which is covalently attached to Ninhydrin. Now, this molecule again reacts another molecule of ninhydrin the same way two times 2 - 2 water so this goes and you get a double bond here. Now this has a lot of resonance it can go here this to the aromatic rings also or it can be the other way around it can stay with the enolate form. And the enolate form will be more aromatic. So, it is because of this structure that gives you the colour. So when ninhydrin reacts with thymine finally you get this compound and this compound is usually deep purple and it is very intense therefore you can use a very trace amount of material even a presence of very trace and quantity of amino acid will show you some colour. This is a test for the primary amines apart from the amino acids if you have any other primary amines with a free NH2 group that will also give you the same taste or the same colour. For the amino acids, if you have proline, proline has a secondary structure as you have seen secondary amine. So, proline will not give the deep purple colour it produces yellow to orange colour. And now for the normal amino acids if you try to measure number one you can visualize the colour that will give you the qualitative test or it can give you also the quantitative test as well you can determine how much quantity of the amino acids are present thereby finding out what is the intensity of the colour. How much colour you are getting and that means if you measure the absorbance of the ninhydrin solution before the start of the test and after the completion of the test then you will see a new peak in the UV region is appearing at around 570-nanometer that depends sometimes on the other water the stuff that is surrounded to it. Around 570-nanometer wavelength, it will show you absorption maximum. And if you calculate the quantity of it and then you can find out exactly how much of amino acid was present. So, the ninhydrin test can be a qualitative test as well as a quantitative test for the detection of the amino acids. You can use the same test for proteins also because the proteins have free amine as side chains lysine especially and of course arginine is also there both of them have free amine as the side chains. So, they can react with ninhydrin can give you the same kind of chemistry and can give you the same colour. So, this is one test for the detection of proteins. second when biochemist or biologists want to isolate a biomolecule then and you want to know whether it is a protein or a DNA or other kinds of biomolecules then you do you can directly measure its UV absorption. So, when you look this is the lambda value this is the absorbance UV absorption this is UV visible absorption spectra. If you look at the structure of amino acids you have aromatic compounds which can absorb UV light chromophore, phenylalanine and tyrosine they absorb a very low region of the UV 200 to 220 in that kind of range which is not a very good wavelength to verify. So, tryptophan essentially that gives you an intense UV absorption sometimes histidine also at around 280 nanometers. So, if you measure you will find a protein sample the absorption of a protein sample or absorbance of a protein sample you will see that around 280 nanometers you have a lambda Max or the absorption maximum. And this is kind of it is not a signature but it is kind of preliminary detection for the proteins once you as a biomolecule if you isolate from a biosource. So, these are the two ways that you use usually to for the preliminary identification of proteins. Now coming to the structure of the proteins so, I will come to the Ramachandran plot just in a bit time. Protein has this skeleton as we have been discussing again and again with the stereochemistry involved in it. Now, this is called the primary structure of the protein which that means simply the sequence of the protein. And then comes how the primary structure of the protein is involved in forming the secondary structure or the tertiary structure of the protein. So secondary structure means that if you mould the protein since they have single bonds if you place them to rotate the single bonds around the protein structures then what are the orientations that you get and what are the short-range interactions that are present within the peptide sequence or the protein sequence. So, secondary structures ideally mean is the short-range interactions and that because of the short-range interactions it has mainly two kinds of geometry that are formed. One is called an alpha helix or the second one is beta-sheet structure. so, these are the side chains. Now if you have for example here I mean as the functional as a sidechain and here if you have acid as the sidechain. So, after certain kind of orientations this I mean free amine and free carboxylate can come close and they can have electrostatic interaction they can have hydrogen bonding that is just for an example. So that will change the structure of the total orientation of the protein and that will make the alpha helix or the beta-sheet structure. So, this is the short-range interaction - or the local interactions that are present within the protein structure. Tertiary structure is the long-range interactions after you get the secondary structure the protein can mould itself again through our long-range introductions less like disulfide bonds and all there are other kinds of interactions hydrophobic interactions and hydrophilic interactions. So that a hydrophobic interaction would repel each other and hydrophilic interactions will bring them closer so that makes the tertiary structure of the protein or the 3d folding structure of the protein we will discuss one by one. So, the secondary structure of protein this is how it looks like it can have at the Alpha helix formation or this is a collagen structure which shows alpha helix formations this is a helix that is formed by the arrangement of the primer acids. Or if your orientation is a zigzag orientation of the basic skeleton then it cannot form the Alpha helix it cannot turn so it does not form the helix instead they can interact with another molecule of the same protein then again another molecule of the same protein and it will make a sheet structure that is known as the beta structure. And again this beta structure can be of two types one is that you have started with N-terminal here this is your C terminal. And the other protein can be on the same direction N terminal and C terminal and then you can see that their backbone this amine and this carboxylic acid they are coming within the hydrogen bond distance these two are coming within the hydrogen bond distance. Like this, there can be hydrogen bonds between the two strands of the same protein and in the same orientation in N terminal to the C terminal. And that can form a hydrogen bond like this similarly another hydrogen bond can be formed in this way and it will make a flat structure this is known as beta-sheet structure. And they are all parallel we call them parallel because they are all from N terminal to C terminal same directions all proteins all protein molecules. Or it can be the other way around one molecule of the protein is N terminal to C terminal other molecules you just make the other way around. This is the C terminal and this is the N terminal this is C again this is N and this is C so it goes this way this one is the antiparallel this one is again parallel. So, these two are anti-parallel these two are antiparallel and that makes them this kind of orientations one of the amino acids is out cannot form the hydrogen bond the others are forming the hydrogen bond in this fashion this one is also not forming. This also makes a flat structure so mainly two kinds of secondary structures so these are all secondary structures short-range interactions as I was saying that is due to the orientation of the peptides how much you can flex the peptide or the protein. And that is where the Ramachandran plot is coming. Ramachandran plot is a way to demonstrate or predict the secondary structure of a protein which form the protein would assume whether it will be an alpha-helix structure or whether it will be a beta-sheet structure that you can visualize or that you can predict using the Ramachandran plot. So, what it does it calculates two angles the rotation around the two angles one is Phi another is Psi. So, it is again given here you have one plane you have the other plane the angle between the two planes is called the dihedral angle and this is a single bond this is the single bond as I have told so it is represented here if I take this as a measure then this is a single bond this is a double bond again this has some double-bond character. So, apart from those two, the bond is a single bond which is termed as Phi and the other one is termed as Psi. So it is NHR and it is RCO these two angles are the difference between the two dihedral angles