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Alistair LOs_Protein Structure and Function_Wk 12 Wine Dark Sea Define primary, secondary and tertiary structure of protein, using haemoglobin as an example. The primary structure The primary structure of a protein is the linear sequence of amino acids. Individual amino acids are joined by peptide bonds, which are amide linkages between the -carboxyl group of one amino acid and the -amino group of another. The free amino end of the protein is the N-terminus while the free carboxyl end is the Cterminus. Secondary Structure The secondary structure of a protein refers to the local structure of the polypeptide chain. This structure is determined by hydrogen bond interactions between the carbonyl oxygen group of one peptide bond and the amide hydrogen of another nearby peptide bond. There are two types of secondary structure, the αhelix and the β-pleated sheet. The α-helix is a rod-like structure with the peptide chain tightly coiled and the side chains of amino acid residues extending outward from the axis of the spiral. Each amide carbonyl group is hydrogen-bonded to the amide hydrogen of a peptide bond that is four residues away along the same chain. There are on average 3.6 amino acid residues per turn of the helix, and the helix winds in a right-handed (clockwise) manner in almost all natural proteins. (The amino acid units on a peptide chain are referred to as amino acid residues. A peptide chain consisting of three amino acid residues is called a tripeptide,) -pleated sheets are formed by two peptide chains running side by side. These are planar/flat. All components of the peptide bond in -pleated sheets and are involved in H-bonding. The individual peptide chains can either run parallel or anti-parallel to one another, the parallel sheet being more stable. -pleated sheets also occur in both fibrous and globular proteins. Tertiary structure The three-dimensional, folded and biologically active conformation of a protein is referred to as its tertiary structure. It consists of complex coiling and folding and gives the protein its final 3D shape. Hydrophobic groups are buried in the centre and interact with each other via hydrophobic interactions away from water. Hydrophilic residues generally accumulate on the proteins surface and interact with each other and water. The tertiary structure is stabilised by amino acid interactions including hydrogen bonds (primary force), covalent disulfide bonds between adjacent cysteine residues (these bonds create permanent loops or coils), ionic interactions and hydrophobic interactions. • Hydrophobic groups are buried in the centre and interact with each other via hydrophobic interactions away from water. • Hydrophilic residues generally accumulate on the proteins surface and interact with each other and water Explain how protein structure relates to function, using the cytochromes, myoglobin and haemoglobin as examples Don’t forget that: Alter the sequence alters the structure which alters the function General Protein Structure Fibrous Protein - linear and have repeated primary sequences and secondary structure – eg. keratin, collagen Globular Protein - globular, roughly spherical in shape, they can have regions of -helix, -sheet and random coil – eg. enzymes and receptors Cytochromes, found in the mitochondrion and endoplasmic reticulum, are proteins that contain heme groups which transport electrons. They are not involved in oxygen transport. In hemoglobin and myoglobin, heme must remain in the ferrous (Fe2+) state; in cytochromes, the heme iron is reversibly reduced and oxidized between the Fe2+ and Fe3+ states as electrons are shuttled from one protein to another. The Fe ion is located right in the middle of the four 5-membered rings (of the haemoglobin) with the pyrrole nitrogen from each ring adjacent to it. The Protein component contains alpha-helices. Myoglobin: Can only bind one molecule of O2 as it only has one heme group. Myoglobin consists of 8 helices arranged into one globular protein. It reversibly binds an oxygen molecule so as to transport it by diffusion from the edge of a muscle cell to a mitochondria where the oxygen will be used. Myoglobin has a higher affinity for O2 than haemoglobin. So the dissociation curve is shifted to left of Hb. That is at the same partial pressure of O2 (pO2) the percent saturation of myoglobin will be higher than that for haemoglobin. Myoglobin has a hyperbolic dissociation curve owing to the fact it binds only one O2. As pO2 decreases the reaction is shifted left to release O2 and if pO2 is increases the reaction is shifted right and O2 stays bound. Note that the high affinity of myoglobin makes it ideal for meeting the O2 demand in the low pO2 conditions found in active muscle. Haemoglobin The solubility of oxygen in blood plasma is very low so it requires a transporter. Haemoglobin transports O2 and CO2 throughout the body within the RBCs (~70%) and is a tetramer of 2 alpha and 2 beta subunits, 22 in adults. It can bind four O2 molecules as it contains 4 haemoglobin subunits each with 1 heme group. Each heme group bind O2 through an Fe2+ ion. The iron atom remains in Fe (II) ferrous oxidation state whether or not the heme is oxygenated (Fe II can be oxidised to Fe III to form methaemoglobin which does not bind O2 – erythrocytes contain methaemoglobin reductase to convert the methaemoglobin that occurs) The heme in each subunit is ligated to one particular His (proximal His). The second His near the Fe ion on the opposite side of the heme (distal His) but not close enough to be a ligand of the Fe2+ ion. Oxygen binds Fe2+ to form oxyHb. There is a conformational change when deoxyHb (T state) oxyHb (R state) Once one O2 is bound it is easier for subsequent O2 to bind. ie. There is an increase in affinity for O2. The positive cooperativity of O2 binding to Hb arises from the effect of the ligand-binding state of one heme on the ligand-binding affinity of another. Therefore the O2-Hb dissociation curve is sigmoidal. Outline the differences in sub-unit structure and function of foetal and adult haemoglobin in terms of affinity for oxygen and 2,3 BPG Sub-unit structure Function & Affinity for O2 Affinity for 2,3bisphosphoglycerate FOETAL (HbF) α2 2 (but once born, β replaces ) GREATER (shifts O2 binding curve LEFT) Affinity needs to be higher than mother’s otherwise won’t be able to ‘steal’ O2 from mother. Mother’s circulation has PO2 of about 50mmHg \foetus has to have greater affinity to take O2 from mother’s Hb. LESSER affinity for 2,3-BPG \can have greater affinity for O2! Lesser affinity ADULT (HbA) α2 β2 (can also get HbA2 which is α2 δ2 if not enough β present eg. thalassaemia) LESSER compared to HbF but balances O2 affinity with ability to RELEASE O2 to tissues and LOAD O2 at the lungs. GREATER affinity for 2,3-BPG. This isn’t a bad thing though because need BPG to (BPG) because globin chain has Ser(-ve charge) instead of β globin chain which has His (+ve charge) \less likely for (–)ve BPG to bind. LESSER Hb’s affinity for O2 so that makes it easier to RELEASE O2. That’s why in high altitude, the body GREATER BPG so that can release more O2 to tissues. 2,3-bisphosphoglycerate (2,3-BPG) reduces haemoglobin’s affinity for O2 2,3-Bisphosphoglycerate (2,3-BPG) is an important by-product of glycolysis in the RBC. 2,3-BPG is a negative allosteric effector of the O2 affinity of Hb. It decreases the O2 affinity of deoxyhemoglobin, promoting the release of O2 in peripheral tissue. BPG binds tightly to deoxy Hb in a 1:1 ratio, but only weakly to oxy Hb so the presence of BPG decreases haemoglobin’s oxygen affinity by keeping it in the deoxy conformation. In arterial blood, where pO2 is 100, Hb is 95% saturated, but in venous blood, where pO2 is 30, it is 55% saturated In passing through capillaries, Hb unloads 40% of its oxygen In the absence of BPG the amount released is decreased, as Hb has a high oxygen affinity and will bind tightly to it In the presence of BPG, more oxygen is released as it decreases the Hb’s affinity for O2. CO2 and BPG independently modulate Hbs O2 affinity In anemia and high altitudes, more BPG is produced, which cannot exit the erythrocyte membrane, and thus causes a decrease in O2 binding affinity, meaning more is unloaded at the capillaries Foetal haemoglobin (HbF) has a higher affinity for O2 than adult haemoglobin (HbA), due to HbF’s lower 2,3bisphosphoglycreate affinity. This is due to the fact the -globin chains of HbF lack some positively charged amino acid residues found in the globin chains that are responsible for binding 2,3-bisphosphoglycerate. Note that HbA and HbF have similar O2 affinities if stripped of 2,3-bisphosphoglycerate but the amino acid differences in the -globin chains of HbF means that in the presence of 2,3-bisphosphoglycerate HbA is at an O2 binding disadvantage. The greater O2 affinity of HbF allows more efficient transport of O2 across the placenta to the RBCs of the foetus as the mother’s blood is less saturated with O2 as it has already passed through part of he circulation as opposed to adults who easily pick up O2 from the lungs. Unloading of O2 to tissues is decreased (harder for O2 to be released from the Hb), and causes a leftward shift in dissociation curve compared with HbA.