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Protein 3D structures, Folding and Denaturation

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Hello everybody and welcome back to the biomolecules, you have a primary structure of the protein that is governed by the sequence of the amino acids and we have seen that if you draw an amino acid sequence in this fashion then there are two different kinds of angles that can be forming. One is for the single bonds so one is this bond which is termed as Phi the other one is this bond which is termed as Psi. So, these two bonds are of course in a different plane and there is an angle that is created between the planes this is called the dihedral angle. And so if you rotate one of them fixing the other then you change the torsional angle and therefore the structure of the protein gets changed. So, this is the primary structure of the protein which essentially means the sequence of the amino acid-specific amino acid sequence. Now the secondary structure of the protein which we have seen that it can either assume alpha helix or beta-sheet that comes from the rotation around the Phi bond or the Psi bond. So, as we have seen here and that you can calculate by using the Ramachandran plot, Ramachandran plot essentially tells you that which rotations of the Phi angle and the Psi angle are allowed. And there is you can put dots or the points for all the rotations if you make a plot of this is the Phi goes from -180 to +180 which makes it 360-degree rotation similarly the Phi and the Psi goes from -180 to +180 that makes it again a 360-degree rotation. So, if you try to rotate them simultaneously or keeping one fixed and rotating the other then you will have a plot like this where these coloured points tell you that which rotations are energetically allowed and those will of course if your density is high then that means those kinds of rotations will give you a stable structure of the protein and that is what we call the secondary structure of the protein which is the Alpha either mostly the alpha helix or the betasheet. There can be different kinds of alpha-helix there can be a different kind of beta-sheet formation. So, if you assume an alpha-helix structure it will look like maybe something like this so where if you rotate one then it will come to such a position that the peptide bond one carbonyl group of the peptide bond and the amine of the other peptide bond may come close and form the hydrogen bond. Similarly, it can be here NH this is C from the backbone and they can come in hydrogen bonding formations that make it Alpha X4 Alpha-helix. Similarly here can be a carbonyl and H and these are the NH and the CEO of the backbone the peptide this NH this CO. Now, this forms the alpha-helix structure. If I consider one of this bond so R if you consider and then what you have is here NH and a CO NH and it goes like this and then you have a CO goes like this so if I make here is your R here is your N and this H see here is your CO it assumes this kind of orientation let us say. So, then if we look at the distance between the NH and the CO so this is around 0-degree angle the distance between the NH and the CO would be like this here and they can come close in proximity and can form maybe hydrogen bonding or may not be I mean they may need to come more closer sometimes. Now technically if I just make a 180-degree rotation then this CO I am rotating around this bond get flipped off you still have the R and you still have the NH like this. Now if the distance between the CO and the NH gets opposite and that creates the change of the secondary structure so here there cannot be a hydrogen bonding between this NH and this CO but it can assume a hydrogen bonding with another CO of a second strand. So if you consider another molecule of the same protein it cannot have a hydrogen bonding with that. Similarly, if you have the other strand they can form hydrogen bonding so that is the difference between the changing of angle by changing the angle you change the geometry of the primary structure that brings you to the secondary structure. So, typically if your Phi and Psi angle as I think I have mentioned it in the last class is around -60 degree then you have an alpha-helix formation. Because it will rotate it and bring it to that level where the NH and the CO can come close in proximity and of course this is governed by the sequence what sequence the peptide has or the protein has. And very commonly if you have alanine, Leucine, methionine, phenylalanine and there are more they typically tend to form alpha-helix kind of structure. And if your Phi versus Psi angle is around -135 degree then that assumes a zigzag structure of the protein and that means they are prone to form beta-sheet structure or beta-sheet as I will call it assembly. Because it happens between two molecules or more than two molecules so this is a supramolecular architecture or supramolecular arrangement of the different protein molecules on the other hand alpha-helix or the hydrogen bonding that exists within the alpha-helix is a more intramolecular process. And beta-sheets are more intermolecular hydrogen bonding and that makes it supramolecular self-assembly. So relatively larger amino acid-like tyrosine, tryptophan and the crowded wants Isoleucine hydrophobic crowded hydrophobic with branched chains isoleucine, valine, cysteine. These kinds of amino acid if they are present in a sequence or if the protein is rich in such sequences then those segments will be more prone to form beta-sheet kind of structure so, within a protein, if you consider a protein within a protein a certain segment of the protein can assume alpha-helix conformation certain other parts of the protein can assume beta-sheet confirmations also. Not necessarily the whole protein will be either alpha helix or beta-sheet. So, that is the secondary structure of the protein alpha-helix or the beta-sheet and this is formed as I mentioned due to the short-range interaction primarily hydrogen bonds. Now once the secondary structure is formed it undergoes further modifications to assume a tertiary structure of the protein which is the functional protein. So, the tertiary structure means is the threedimensional structures or the folded structure of the protein which comes from the coiled formation. So the protein will further mould into a fold its form or a coiled form that is known as the tertiary structure. And this is because of relatively long-range a bonding interaction that assumes the tertiary structures. I will just show you in a brief period how the interactions will take place? What are the long-range introductions that happen? This is mainly for example that if you have a secondary structure which is the alpha-helix here. And then if you have a polar group or let us say an amine functional group here and then a carboxylate functional group sidechain in this case and then they want to come in close in proximity because of the electrostatic interactions. The amine group is positively charged and the acid group is negatively charged so they want to come closer together. So, that will force this helix to go further fold so that the negative charge and the positive charge can come closer and form an electrostatic bond. Similarly, there are other types of interactions that I will just briefly explain very quick. So, these are the long-range arranged bonding interactions that form a tertiary structure of the protein. Now, apart from the secondary structure, there is another structure that some proteins have that is known as the quaternary structure. Quaternary structure is basically if a protein has many subunits more than one unit of a protein there are many other units we call them subunits or they are called as oligomeric proteins. So, those different subunits of the protein are interconnected of course so that way they are interconnected and form our total structure is known as the quaternary structure. so, it is the quaternary structure in the assembly of the subunits of a protein. So, as you know that when a protein is formed, protein is usually biosynthesized in our cellular systems in the living cells. How is it will be synthesized all the details will have a separate chapter for it that it comes from our genetic code that is DNA and then it goes to the formation of a messenger RNA or known as mRNA and from mRNA it is transferred to tRNA and then the tRNA synthesizes the protein. So, when there is the synthesis of the protein, of course, it synthesizes the covalently linked primary structures the sequence of the protein. So, once our sequence of the protein is synthesized then depending upon its sequence it will automatically resume or assume the secondary structure and then the secondary structure will go to the tertiary structures that are the functional protein. So, once a tertiary structure of a protein is formed then only it can do our biological transformation or it will have up certain biological activity. And, again and again, I am repeating that what kind of tertiary structure a protein will assume is always dictated by the primary structure of the protein that is what sequence it has. Now there are a lot of studies that have been done or still been carried out to understand how the protein is folded into that kind of specific structure why not anything else why not a slightly different type of three-dimensional structure possible for the protein. Similarly how you can unfold a protein from the tertiary structure to its primary structure. And to study the dynamics of those all these processes. So, we call it a typically a denaturation when a protein is unfolded that means you are breaking all the interactions that are present in the tertiary structure and then bringing it back to the kind of primary structure so that is the process is known as as the denaturation of the protein or you can also call it unfolding of a protein. So, the moment you unfold a protein or you force a protein to denature its threedimensional tertiary structure is disrupted and therefore the protein cannot function anymore this is called the inactive protein. And we will see just in a brief time that folding of a protein from this primary sequence to the tertiary structure is a spontaneous process under physiological condition and of course the primary structure determines the tertiary structure of the protein. So, now what kind of interactions are present within the tertiary structure of a protein if you understand that then only you can think of breaking those interactions and make and unfold or denature a protein. You can find a suitable way how to denature a protein or how to unfold a protein and then can find out again methods to study the dynamics of those processes. So, if I take an example of a tertiary structure just I am just drawing anything arbitrary like this so what are the forces that makes a tertiary structure or what are the bonding interactions that force a protein to assume a tertiary structure. One of them is, of course, the interactions of the sidechains. Sidechains can be of very different types as we have seen we have 20 amino acids with very different functional groups very different properties. So, depending upon the sidechains the interactions can vary also. So, one is if you have the electrostatic interactions, electrostatic interactions what does that mean? So when you have seen that secondary structure you have seen the hydrogen bonding so were present within the Strand for alpha-helix for beta-sheet it was between the molecules. So here further for the sidechain, if I consider for example I consider a lysine. Lysine as amine sidechain there are 4 CH2 groups I am not drawing them and there is amine here and this amine assumes as you have seen in two it is positive charge form. Now if you have closed in the opposite direction somewhere if you make it a flat structure you will see the distance between this lysine and this residue here is very large you have to go all the way here. But because of such interactions like if you have aspartic acid for example aspartic acid is CH2 and then you have a CO then you have OH and this OH, of course, exists in O minus. So now you have a positive charge you have a negative charge so if in the secondary structure if there is a chance that this lysine and the aspartic acid somewhere else are to some extent close then to form a better bond to form electrostatic interactions between the positive charge and the negative charge it will force the strand to mould more and bring the positive charge and negative charge within the bonding interactions. So, that forms the tertiary structure and of course, this is a long-range interaction as I am saying because if you look at the distance between this and this is quite long. But the spatial distribution or the special arrangement forces these two residues to come closer together and that holds the protein. So, one reason for assuming the tertiary structure is electrostatic interaction. Second, if there is an electrostatic interaction and then there will be the opposite one the hydrophobic interactions also. Hydrophobic interactions mean so when a protein is in a solution typically in within the biological cells as I have said that it has a pH of 7.4 so more or less and it is an aqueous medium. So, protein is in an aqueous medium in a pH of 7.4 roughly. So, water molecules are surrounding it and hydrophobic side chains of the amino acids they repel water so they would not want to be exposed towards the water, and they will tend to go away from water and height. So, typically that happens here so if you have multiple numbers of hydrophobic residues or hydrophobic sidechains, for example, the valine in isoleucine and may be fitted phenylalanine and so on so they would want to go or hide away from water and they will form a core. So, if you have for example of valine here I am just making another coil, valine here CH3 CH3 and maybe another valine very close here. If you have phenylalanine here so they will all try to gather themselves to form a hydrophobic core it says called the Van der Waals interactions or hydrophobic interactions. So, all the water molecules outside here surrounding the protein. So, they will Psi away from the protein from the water and want to go inside the interior of the protein. And once they go inside the protein they have the hydrophobic molecules also have the hydrophobic interactions within them so that is a stability factor. So second is hydrophobic interactions a third one very common is the formation of the disulfide bond. So, if you have a cysteine so this is cysteine within one protein molecule and let us say there is another system may be within the same protein or can be in a different protein and a different protein molecule so CH2 and NH. So, these two if you keep if you take a cysteine and keep it in the air so it will immediately undergo oxidation so redox reaction that will form CH2 S S CH2 and here is this CH2. And here is the rest of it CO and the NH and the rest of it, so it will form a disulfide bond and that is a covalent bond so a quite strong bond. So, cysteine-cysteine residues if they are in close proximity that will tend to form a disulfide bond if you keep it in the air under aerial condition or anaerobic condition. So, and that will further force the helix or the beta-sheet to mould it and bring them closer so that they can form the covalent bond. So, here if you have a self SH residue or the cysteine residue on the same strand somewhere else if you have another system let you do and if they are in open-chain so they will fold themselves and form the disulfide bond that is the driving force. The formation of the disulfide bond is a driving force for moulding the structure and there are other interactions in the next slide I will show you with proper examples. So these are primarily 3 types of interactions that dictate the formation of the tertiary structure. So, I will give you an example of how the disulfide bond is formed in protein. So, very well-studied protein is bovine insulin you know insulin is a medicine that is used to treat the diabetic patients right it is an enzyme. So, bovine insulin was the insulin which used to be used as the medicine to treat such diabetic patients. Nowadays we can manufacture insulin from human sources itself after the discovery of the recombinant DNA techniques. So, bovine insulin is a very well studied protein and that shows multiple numbers of disulfide bonds. So, it has two chains it is called a chain and that has a sequence I will just write I am writing the part of the sequence glutamic acid then glutamine then you have a cysteine another cysteine, alanine, Serine, valine assistance then I said in and it goes and there is another cysteine at the position 20. So, this cysteine position is 6 this is 7 position and this is 20th for the opposition of the cysteine. Now this cysteine has an SH residue and they can form the disulfide bond that locks the protein structure. Similarly, the second cysteine I am sorry cysteine alone insulin valine cysteine I'm sorry for here there is SH and this is and this is can form the disulfide bond. And therefore the protein structure has two moulds or has two coils so that these two can come within the bonding distance bonding interactions. Similarly, this has an SH and in the next chain of the protein molecule of insulin known as the B chain in this, there is another cysteine which comes closer to its opposite. So, before it has a leucine then histidine and then glutamine and so on. so, this is also the s7th position of it. And then here there is another cysteine and this position is 19. So, this system has tile and they form disulfide bond very quickly. Similarly, the s of this the s of this they form disulfide bond very quickly. So, that overall structure has are many disulfide bonds and that is the driving force for the tertiary structure of the bovine insulin. So, here I have made a schematic presentation of how different interactions are present that makes our tertiary structure of the protein. So, this is, for example, this is the secondary structure of the protein and it gets down to the tertiary structure of the protein which is the coiled form of the folded form. So, basic laws of chemistry that drive the protein folding are first is the hydrophobic sidechains that have been termed given the colour like black, for example, this and this they are the hydrophobic side chains as I mentioned valine isoleucine leucine phenylalanine all those they do not have our functional groups or polar sidechains. So, they are termed as black here they will be buried on the inside of the globular protein where they are hidden from polar water molecules. Water is polar and they are surrounding the protein molecules as I have explained so these things when they form the tertiary structure they want to hide away from water and forms this core kind of structures. So, that is the driving force for the protein to fold in this fashion. Second is the charged side chains means it can be up to charge on it is positive charge other is the negative charge is for the acid residues like the aspartic acid and glutamic acid. The positive charges coming from the amine residues, for example, the lysine and arginine both have a mean sidechain. So, blue and the red are marked for positive and negative sidechains. So, they will be on the surface of the proteins. Because since they are charged species they would want to interact with water molecules also because the water can reach them is polar or and of course, the charged species are also polar so they primarily would be exposed towards the water. And of course, they will have the interactions within them bonding interactions or electrostatic interactions within them positive and negative. Polar side chains other than the charged ones which have polar functional groups will be again on the surface of the protein they would be on the surface. So that is represented by green dots they would be on the surface of the protein because that is the part where they are exposed to the water molecule. So, they can interact with a water molecule and get solvated that is a very effective stability factor the solvation energy when a molecule solvated or surrounded by water molecules and that is a highly energetically favourable process. So, the polar sidechains will be on the surface of the protein and they can form the hydrogen bond with the water that is the solvation factor. Fourth is the cysteine side chains cysteine is tough I have given it as purple colour here for our bond formation SS bond formation that often interacts with each other to form covalent disulfide bonds that stabilize the protein structure. So, these are the factors that are responsible for the formation of the tertiary structure of a protein. And obviously, now you can see how the primary sequence or how the primary structure of the protein the sequence of the amino acids are dictating the folding of the protein depending upon their sidechains. So, a classic study of how the protein folds and unfold was done by Professor Anfinsen in this is called Anfinsen’s experiment or Anfinsen’s protein denaturation and folding. So, Anfinsen received Nobel Prize in 1972 in Chemistry he has been working for a very long time on the enzyme ribonuclease Rnase. He was one of the key persons who has worked a lot on RNase that is ribonuclease and a part of his research was, of course, to see how the tertiary structure is formed in the protein. Why it is such why does it assume such specific structure how you can unfold a protein and can inactivate a protein. So, he has done a complete study and dynamic study for the protein denaturation and refolding. So, it is a sequence of process and that first studies the folding process. what he has done is first he tried to denature the protein and how can you denature the protein and then refold the protein again. So we have seen what are the forces that are involved in the tertiary structure. Now if you want to denature a protein then you have to break all those interactions. And therefore now you can find a way or think of a way how can you denature though or how can you break such kind of interactions then only you can denature the protein. One of them is, of course, heat you can heat the protein high up and then break all the non-covalent interactions but it still has the problem that sometimes the protein degrades by itself or there are other bonds as you will see that especially the disulfide bonds and other which are hard to cleave by heat. But there are other regions which you can cleverly use to clip those bonds. So, denaturation of a protein means the loss of tertiary structure once. The tertiary structure is lost it will lose its activity. So, what Anfinsen found out that if you use urea then you can denature a protein. Urea is not very simple reagent, of course, is a reagent that denatures the protein. It disrupts all the non-covalent bonding interactions within present within the protein and therefore you break the native tertiary structure as well as the quaternary structure. But after denaturation with the urea, you still have the disulfide bonds left which you have to cleave and therefore the second reagent is coming which a mercaptoethanol. It is a very simple reagent structure is very simple what it does is that it is anti redox I mean it changes it undo the process of the disulfide bond formation. So, when you have the cysteine here and cysteine there is a protein they formed a disulfide bond. Now if you treat these with beta-mercaptoethanol this will reverse the process it will make plus two individual cysteines by cleaving the disulfide bond. And in the process this molecule itself so this is also and this has also a thymine bond two of these molecules will undergo disulfide bond formation OH S CH2 CH2 OH. So, this will form the disulfide bond and undo this process. so, it is a redox reaction as I have said so this will be oxidized and the protein will be reduced to individual systems. Therefore you can break the cysteine-cysteine disulfide bonds also. Here is how the process goes on it is done in two steps the first step is the treatment of the protein that is, in this case, RNase with H molar of urea and function has found out that the concentration of urea is optimum if you use it on 6 to 8 molar urea is ideal for denaturation of the protein. So, that is the optimum condition to denature the protein. So, once you use urea 6 to 8 molar then it breaks all the non-covalent bonding interactions such as the hydrogen bonding that were present. Hydrogen bonding so is present within the secondary structure also as I mentioned as well as in the tertiary structures with the water molecule within them within the molecule as well as the polar if you have the polar sidechains and they form hydrogen bonding with water also. So, lots of hydrogen bonding will be present within for the tertiary structure of the protein itself as well as the secondary structure of the protein. So, urea will break all the hydrogen bonding interactions at the same time it will break all the charged interactions all the other Vander Waals interactions and the electrostatic interactions also like the charge species lysine with the aspartic acid those kinds of interactions will all be broken by using Urea. But still, once you destroy all the short-range interactions or the longrange bonding interactions you still have written the disulfide bonds. So, therefore after treating with urea it partially denatures the native tertiary structure. Second, as I said is a treatment with a beta-mercaptoethanol it is a redox reducing reagent that will oxidize itself and reduce the disulfide bond back to the cysteine. So, once you treat this with the beta-mercaptoethanol then you have completely disrupted the tertiary structure of the protein and that will lose the native structure therefore complete denaturation will happen. And once you denature the protein that then the protein or in this case the RNase is completely inactivated it cannot do its biological function that is what Anfinsen has found out and have studied. So, after this treatment or of this two consecutive process and Anfinsen has found out that RNase the enzyme RNase does not retain its biological activity. The question is can you refold the protein can you make the protein functional again. So, that is what is very interesting study actually once you denature the protein it can assume its activity biological function again which means the unfolding structure or unfolded structure and the folded structure of the protein do they exist in equilibrium. So, that is what he has also studied. Now to refold the protein what you have to do so you have urea in it by using urea you have disrupted all the short-range interactions or all the weak interactions weak bonding interactions non-covalent interactions I mean.