Denaturation and Renaturation

In the last lecture , we talked about protein structure ( primary ,secondary and tertiary ). Before discussing quaternary structure ,we are going to talk about properties of proteins . Denaturation and renaturation (one of the chemical properties) denaturation is the process in which the native conformation (3D shape) is disrupted due to some changes in the surrounding environment , or the application of certain conditions around it . Denaturation involves the breaking of the non - covalent bonds as well as disruption of disulfide bridges (important covalent bond for stabilizing the folded protein). Complete disruption of tertiary structure is achieved by reduction of disulfide bonds in a protein (Figure 1). When the proteins lose these non-covalent interactions and disulfide bridges , they are going to become in an unfolded state and the protein will lose its properties such as activity and become insoluble in water. How can we denature proteins ? by using denaturing agents such as: 1. Heat is one of the denaturing agents which can disrupt low-energy van der waals forces in proteins . For example: when you boil an egg , the change between the jelly like material that made up the proteins inside an egg becomes in the solid state and they become whitish in color , these differences are due to the denaturation that happened to the protein content of the egg (and the denaturation is caused by the exposure to heat). 2. Extreme changes of PH( at either high or low ) : if there was a big change in PH value , there would be disruption and interference with the formation of different types of noncovalent interactions specifically the electrostatic interactions and hydrogen bonding because the changes in the protonation and deprotonation particularly for acidic and basic 1 amino acids , so they would accept or donate their protons and become with a different charge than the original one at physiological conditions . 3. Using some chemical detergents like triton x-100(nonionic, uncharged) and sodium dodecyl sulfate (SDS, anionic , charged ). note 1: Detergents tend to disrupt hydrophobic interactions . note 2: If a detergent is charged (like SDS), it can also disrupt electrostatic interactions within the protein . 4. Other reagents , such as urea and guanidine hydrochloride disrupt hydrogen bonding and hydrophobic interactions . 5. Reducing agents such as β-Mercaptoethanol (βME) and dithiothreitol (DTT) are used to reduce disulfide bridges to two sulfhydryl groups , and break down the connection between different cysteines along the polypeptide chains . Remember: cysteine is one of the few amino acids that contains sulfur. Can we go back to the functional form and 3D shape ? Yes we can( for some proteins but not all of them) and this process is called Renaturation. Renaturation is the process in which the native conformation of a protein is re-acquired. Y can ge he egg back to its original shape because the proteins of the egg can renaturate to their original functional state (3D Shape). When denaturing conditions are removed like reducing agents or exposing proteins to oxidizing agent , Some of them and specifically those dependent on disulfide bonding can renaturate and their activity is recovered . (Figure 2) renaturation can occur spontaneously disulfide bonds are formed correctly . and 2 Factors that determine protein structure 1. The least amount of energy needed to stabilize the protein .This is determined by: The most important factor is the amino acid sequence (the primary structure)mainly the internal residues , because the content of amino acids and the difference in R-chains ,their ability to interact with each other, the distances between them, the orientation of these R-groups ,etc. The proper angles between the amino acids, specifically between R-groups . 2. The different sets of week non-covalent bonds that form between mainly the R-groups. 3. Non-protein molecules like prosthetic groups of different types, metal ions, heme groups , sugar and lipid component . Can an unfolded protein re-fold? If a protein is unfolded, it can refold to its correct structure by placing the S-S bonds in the right orientation (adjacent to each other prior to formation),then the correct SS bond are formed . This is particularly true for small proteins . Note : for denaturation that happened due to disruption of non-covalent interaction, it will be harder to go back , so it is mostly irreversible as a process but it might be in some instances reversible . What is the problem in misfolded proteins ? When ein d n f ld c ec l hei in e nal hydrophobic regions become exposed and interact with other hydrophobic regions on other molecules ,and form aggregates . Once we lose the 3D shape we are going to lose function, proteins in (figure 3-A) still partially folded and can be exposed to certain conditions where they can continue the folding process and reach the final shape that is functional . Nonpolar amino acids are going to be on the inner side of the protein.(normal situation). 3 Proteins in (Figure 3-B) have a problem in folding ,therefore the proteins may stay partially folded or completely unfolded ,nonpolar amino acids that should go to the inside of proteins stay on the outside surface of the proteins, and this is unstable specifically when we talk about soluble proteins that are exposed to aqueous environment (like cytosol, ECM and bloodstream). To stabilize these molecules ,these non-polar regions are going to interact with each other to hide themselves from the aqueous environment surrounding them ,and start to form different types of aggregates ( Dimeric < Trimeric < Fibril ). How would aggregates affect the function of the cell ? As we have seen before in the sickle cell anemia the hemoglobin started to accumulate and clump to hide these non polar amino acids ,and that changed the shape of cell and reflected on the function of the whole cell(you can extrapolate this example on other proteins with misfolding). Also aggregation may reach to the point of forming fibril structure or amyloid like what we can see in Alzheimer disease for example (we are going to talk about Alzheimer in page 8 ) The problem with aggregates is that they may change the shape of the cell, and affect the physical properties of the cell itself like the rigidity and bendability . Also, they can be toxic to these cells and reduce the ability to perform their functions properly . 4 How can we solve the problem of misfolding (or its affect which is aggregation) ? By using chaperones ,these proteins bind to polypeptide chains and help them fold with the most energetically favorable bathway. Chaperons also prevent the hydrophobic regions in newly synthesized protein chains from associating with each other to form protein aggregates (Figure 4). Chaperons can help these proteins while they are being synthesized or after synthesis is done to fold into the proper 3D shape ,even if the proteins failed to fold properly . Chaperons may unwind the proteins and refold them properly b if he d n reach the final functional form and correct folding, they may target them for degradation ,and this is for the favor of the cell because if they stay with an abnormal function or no function this may result in different types of diseases . Outcome of protein misfolding Partly folded or misfolded polypeptides or fragments may sometimes associate with similar chains to form aggregates . Aggregates vary in size from soluble dimers and trimers up to insoluble fibrillar structure (amyloid)(like what we can see in Alzheimer ). Both soluble and insoluble aggregates can be toxic to cells . Note: if he cha e n didn k eall ell and c ec ed he misfolded or unfolded proteins into the correct shape , this result in the formation of different types of aggregates that may reach to the point of forming fibril structure . Another examples related to protein misfolding prion disease: they are proteins that can infect each other and convert normal ones into an abnormal and they cause the : mad cow disease (in cow). Creutzfledt-jacob(in humans ) Scrapie (in sheep). it is a form of encephalopathy(problem in the neural system ) . o Pathological conditions(like encephalopathy ) can result if a brain protein known as prion protein (prp) is misfolded into an incorrect form called prpsc .(Figure 5-B) o prpc has a lot of α-helical conformation, but prpsc has more β strands forming aggregates . In (Figure 6-B) the abnormal prion proteins start to accumulate forming dimers of different types (heterodimers) and dimers of the same types(homodimer) And they keep accumulating to form amyloid aggregates . Note: amyloid aggregates disrupt certain types of nervous cells . o In prion diseases, the disease is caused by a transmissible agent .So, there has to be some sort of transfer of the abnormal misfolded prion from a cow to a human(for e am le and hen nce i in ide he memb ane f he h man cell , the abnormally folded protein is going to start infecting the normal proteins in the membrane of these cells, thus they become abnormal and start to affect the other normal proteins until they disrupt the function of these cells. o Abnormal protein can be acquired by : o Infection . o Inheritance . o Spontaneously . Let s take another example where we have aggregates of miss-folded proteins : Al hei e di ea e This disease is not transmissible between individuals it s not an infectious disease ). Family s history may increase the probability of getting this disease. the age of the onset (the age when we start to notice the symptoms of the disease) might be earlier in some families in which this disease runs. In this disease there would be accumulation of certain types of aggregates or plaques which are called (amyloid plaques), it s not normal for these palques to be present in the interstitial fluid between neurons. These plaques are actually made of amyloid peptides and also a protein called (tau), so they start damaging the neurons and this results in alzheimer s disease. These aggregations cause Alzheimer Let s see how the aggregation and formation of these beta amyloids happens this is the protein (APP), in the normal person it can be cleaved by alpha-secretase enzyme. This enzyme cleaves the APP protein in the middle of this peptide (A-beta) so APP protein will be divided to 2 fragmets and these 2 fragments can be released easily to perform other functions. Whereas whenever there is a problem with the cleavage itself, so normal cleavage can t be 8 performed due to a mutation in the APP protein that would affect the function of betasacratase for example and this causes cleavage to happen in other sites (other than the normal position of cleaving) so now we are cleaving beside A-beta peptide as in the picture above and we still have this A-beta peptide undamaged so it will be released out of the membrane to the extracellular space. and since this A-beta peptide is inserted in the membrane as you can see in the picture we would expect it to contain hydrophopic amino acid (because the membrane from inside is hydrophopic), and these hydrophopic amino acid can t be stable once they are outside of the membrane (because they are hydrophobic and the solution outside is hydrophilic). So because it s made of hydrophobic amino acids it will start to accumulate and aggregate due to the forces of hydrophobic interactions that try to isolate these hydophobic amino acid from the surrounding aqueous environment leading to the fomation of beta amyloid aggregates and that s what causes alzheimar s disease. Quaternary structure The quaternary structure of a protein is the association of several protein chains or subunits into a closely packed arrangement,where each subunit has it s own primary, secondary and tertiary structure. Some proteins are composed of more than one polypeptide chain, this means that they have more than one N-terminus and more than one C-terminus, each of these polypeptide chains is considered as a subunit and the association of these subunits forms the quaternary structure. The proteins that are composed of one subunit are monomers and they don t actually have a quaternary structure. Proteins made of Two subunits = dimers Three subunits = trimers Four subunits = tetramers etc So the quaternary structure is the arrangement of all these subunits relative to each other in the 3D space and the type of interactions that connect them together to form one unit 9 that is a functional protein. *Not all proteins have this level of protein strucure (the quaternary). The subunits making a protein may be the same polypeptides or different ones. In case we have identical subunits we call them homooligomers (homo=same). In case we have different subunits we call them heterooligomers (hetero=different). Examples: homodimers (the simplest one), hetrotrimers, homotetramers depending on the number of subunits present in a protein . How are the subunits connected ? Sometimes subunits are disulfide-bonded together (disulfide bridges between subunits) Other times, non covalent bonds stabilize interactions between subunits. For examble the hemoglobin protein, it s composed of subunits 2 alpha and 2 beta, the connection between them can be achieved by different ways, non covalent interactions and disulfide bonds. *Hemoglobin is a heterotetramer. *Disulfide bonds are considered covalent bonds. *There is no peptide bonds between subunits. *The most common covalent bond between subunits is the disulfide bond. In other examples like in antibodies or immunoglobulins, they have 2 light and 2 heavy chains and they are connected by a covalent bond that is actually a disulfide bridge between the cysteine amino acids (because they contain sulfer atoms). So the connection might be through disulfide bridges or noncovalent interactions. 10 Complex protein structures Halo- and apo-proteins As we mentioned before in the tertiary structure that prosthetic groups are added to some proteins to reach the final functional form, for example: glycoproteins can t do their function without adding the sugar component to the protein, same thing for hemoglobin as it will not function without the heme groups. This addition of prosthetic groups can be preformed at the tertiary structure of the protein. Proteins that are linked (Conjugated) to non-protein molecules (prosthetic molecules) -like heme group for example- are called Holo-proteins, also called (cunjugated proteins). Once we remove that non protein component out of them they are called Apo-protein. So Apo-protein is just a protein that is not connected to any prosthetic groups, once we add a prosthetic group they are called Holo protein. Glycoproteins are an example of Holo-proteins and the suger component is covalently linked to the protein (N-linked glycosylation and O-linked glycosylation for proteins). This is the site of the addition of carbohydrate structures to the glycoprotein. * they are added to the heavy chains* **Antibodies are glycoproteins** 11 Classes of glycoproteins 1-N-linked sugers 2-O-linked sugers Remember: N-linked glycosylation is when we add an oligosaccharide to the amide nitrogen of an asparagine (Asn) residue of a protein. O-linked glycosylation is when we add an oligosaccharide to the oxygen atom of serine (ser) or threonine (Thr) or occasionally hydroxylysine (Hyl) residues in a protein. Other examples on Holo-proteins are lipoproteins (we discussed it with Dr.Mamoun in the lipid section). Lipoproteins have both lipid molecules and proteins. Another example of Holo-proteins is phosphoprotein, it s formed when a protein gets phosphorylated. Proteins can alternate between Holo- and Apo-protein states like the proteins that gets phosphorylated and unphosphorlyated. This protein has no phosphate group added, and it s functional. If it s an enzyme, Phosphorylating it may activate it or inactivate it depending on the enzyme itself. It gets phosphorylated by a kinase enzyme (protein kinase). Some proteins are always conjugated to their prosthetic groups and they can t function without these groups. 12