Heamoglobin, membrane proteins and fibrous protiens.docx

Structure of myoglobin and haemoglobin Both contain haem prosthetic groups. Oxygen can bind to iron within the haem group. Both exists in a deoxygenated and oxygenated form. Haem group is planar. Myoglobin Predominately alpha helices Has 8 major helical regions. Has a compact tertiary structure....

Structure of myoglobin and haemoglobin Both contain haem prosthetic groups. Oxygen can bind to iron within the haem group. Both exists in a deoxygenated and oxygenated form. Haem group is planar. Myoglobin Predominately alpha helices Has 8 major helical regions. Has a compact tertiary structure. Monomeric Haemoglobin Tetramer 2 alpha subunits 2 beta subunits Each subunit is folded similarly to myoglobin. Haem groups are apart from each other. Relationship between oxygen concentration and binding to haemoglobin. Haemoglobin becomes saturated at high concentration of oxygen and vice versa at low concentrations of oxygen. Different concentrations of oxygen are present in the lung’s vs the tissues. In the lungs, the haemoglobin gets fully saturated with oxygen due to having a higher concentration of oxygen in the lungs. In tissues, there is a lower concentration of oxygen, the saturation/affinity decreases which allows the haemoglobin to release oxygen into the tissues. Related to affinity. Affinity refers to how tightly 2 molecules interact or bind. Myoglobin has a much higher affinity compared to haemoglobin meaning myoglobin would not be effective transporter of oxygen because it binds to oxygen so tightly, it would still hold onto the oxygen when it needs to be released. Myoglobin is used as a storage for oxygen and designed to release oxygen when very limited. In contrast- Haemoglobin has a lower affinity which enables it to release it to the tissues. . Haemoglobin has this S shape (sigmoid) graph which is due to cooperativity. What happens when oxygen binds to haemoglobin? Binding of oxygen induces a confirmational change and forms oxyhaemoglobin. Cooperativity between subunits. The deoxygenated form is referred to as T state. The oxygenated form is referred to as R state. The R state increases the affinity for oxygen because binding of oxygen to one subunit increases affinity for oxygen at neighbouring subunits. Allostery- binding of a ligand to one site of protein which affects the binding properties of another site on the same protein. Cooperativity- occurs mainly in multimeric assemblies. The binding of one ligand affects their affinities of anu remaining unfilled ligand binding sites. Gives a sigmoidal binding curve. Binding of oxygen to haemoglobin is both Allostery and Cooperativity Why purified haemoglobin has a higher affinity for oxygen than it does within red blood cells? When haemoglobin was purified, 2,3-bisphosphoglycerate (BPG) was removed that’s critical to the function. BPG is present in the red blood cells at approx same concentration as haemoglobin. It is critical for haemoglobin to be an efficient oxygen transporter. BPG only binds to deoxygenated haemoglobin and one molecule of BPG binds per haemoglobin tetramer. BPG is highly negatively charged, and the surrounding is positively charged. BPG binding cross links the two beta subunits by electrostatic interactions. BPG stabilises the deoxygenated form. BPG is an allosteric effector. Foetal haemoglobin Has a higher affinity for oxygen hence the curve is more towards the right. Foetal haemoglobin has different composition- has 2 alpha and gamma chains. Gamma chain is 72% identical to beta chain but there is 1 big difference which is the substitution of His143 residue with a serine. His143 residue is an important residue for binding BPG. Therefore, Foetal haemoglobin binds to BPG with a lower affinity than adults’ haemoglobin does. Lower affinity for BPG= higher affinity for oxygen Sickle cell anaemia- genetic disease RBC sickle upon deoxygenation of haemoglobin. Sickle cells are fragile and can rupture easily therefore can cause anaemia where oxygen isn’t transported around efficiently. Can be life-threatening. Caused by a single nucleotide mutation in the gene that encodes haemoglobin beta subunit. Results in substitution, Glu6 for valine. Valine is hydrophobic whereas Glutamate is hydrophilic. Upon deoxygenation of haemoglobin, a hydrophobic patch gets exposed made of Phe85, Val88. This is normal but now due to substitution of Glu6 for Valine, 2 hydrophobic patches are now exposed instead one 1 which are the Val6 patch, Phe85 patch and Val88 patch. The problem is when the oxyhaemoglobin sickle cell (with valine on the surface) and deoxyhaemoglobin sickle cell (with the hydrophobic patch). Those come together and forms a long chain and forms insoluble fibre which then causes the RBC to sickle. Bohr effect Respiring tissues produce large amounts of CO2 and H+ To release oxygen where it’s needed, haemoglobin has evolved to respond accordingly. Increase in H+decrease in pH. CO2 + H2O--H+ HCO3- Catalysed by carbonic hydrase. Effect of decrease in pH It allows for greater release of oxygen as the affinity decreases. What causes this? His146 can be protonated when the pH is lower- allows electrostatic interactions which stabilises the deoxygenated T state of haemoglobin so decreases affinity for oxygen. CO2 also directly affects haemoglobin. Even lower affinity for oxygen therefore more oxygen can get released. CO2 reacts with N-terminal amino groups to form carbamate groups which are negatively charged. Electrostatic interactions occur that stabilises the deoxygenated T state of haemoglobin so further decrease in affinity. Therefore 3 things that decrease the haemoglobins affinity for oxygen thus it releases oxygen better in places where it’s needed such as tissues are: Binding of BPG Decreased pH Increases CO2 CO poisoning Colourless and odourless gas Binds to haemoglobin at the same site as oxygen. CO has approx 200x more affinity than oxygen so even small amounts of CO displace oxygen. If CO is bound to one subunit and the rest of 3 are bound to oxygen, it increases oxygens affinity so high that it never releases oxygen- hypoxia. Fibrous proteins Fibrous proteins are insoluble in water. Consists of single type of secondary structure Simple tertiary structure Provides structural support and shape for cells and tissues. Examples Alpha-keratin found in hair and nails etc. Formed from 2 long alpha helices that wrap around each other to form a left-handed coiled coil. (alpha helices are right-handed so 2 of them together makes a left-handed one). Very stable and can be long. Contains regions of amino acids containing imperfect heptad repeats. Every 7th residue is leucine (hydrophobic amino acid) 2 leucine’s matches up from both chains when they come together and stick to each other due to being hydrophobic. Structure stabilised by London forces and ionic forces. Can also have disulphide bond between the helices. Number of disulphide bonds effect properties such as hair has less of the bonds as its flexible. Silk fibroin Produced by spiders etc. Predominantly made of beta-sheets. Beta-sheets stack together. Rich in Ala and Gly because they have smallest side chains- important because it allows close packing and interlocking of beta sheets. Stabilised by hydrogen bonds and London forces. Collagen Main component of bones, teeth, skin etc. Has a unique secondary structure Forms a left-handed helix. Only has 3 amino acid per turn so tighter. 3 of these polypeptides twist together in a right-handed coiled coil. Every 3rd residue is Glycine. Contains a lot of prolines and hydroxylated prolines. Can get both hydroxylated proline and lysine residues within collagen. Proline is important for folding of collagen because it is the only amino acid where side chain bends around and covalently attaches to amino group. Causes a kink in chain. It allows the chain to fold up into final shape. Glycine has the smallest side chain so it fits in the centre of the coiled coil which allows tight packing. Collagen fibrils 3 polypeptides twist together and align in a staggered formation. They are cross-linked for strength. Membrane proteins Functions include: Cell-cell recognition Transport Signal transduction Can be peripheral or integral. Monotopic Polytopic Peripheral Bound by electrostatic interactions and hydrogen bonding to lipid head groups. Types of peripheral membrane proteins Amphipathic alpha helix Hydrophobic loop Lipidation Electrostatics Lipids are covalently attached to proteins, these insert into the membrane, anchoring the protein. (hydrophobic as they insert into the membrane). Most of transmembrane protein are alpha helical. But can also be beta-barrels. Hydropathy plots Can predict the presence of transmembrane alpha helices using algorightms that detect hydrophobic amino acids. Side chain localisation Hydrophobic side chains protude into bilayer core Charged and polar side chains are in regions outside of the membrane Tyrosine and tryptophans residues are often located at the water lipid interface Membrane proteins can be channels or transporters therefore may have hydrophillic residues in the translocation pore. Membrane proteins are not solubale in water hence they are hard to stay as they are often embedded in the membrane. Often detegernets are used to solubilise membrane protein Detergents have hydrophillic and hydrophobic regions. It then forms micelles around the hydrophobic regions of the protein and makes them soluable in water.