Topic 12 - Protein Function, Regulation + Oxygen T a7fd2b3139084daeb6f19ea8b406d1ab.pdf

🆎 Topic 12 - Protein Function, Regulation + Oxygen Transport Explain the physiological roles of haemoglobin. –What do they do? Haemoglobin is a tetrameric haem protein that transports oxygen....

🆎 Topic 12 - Protein Function, Regulation + Oxygen Transport Explain the physiological roles of haemoglobin. –What do they do? Haemoglobin is a tetrameric haem protein that transports oxygen. Globular Structure - two alpha polypeptide chains and two beta polypeptide chains forming Haemoglobin has low oxygen affinity, high dissociation. each subunit has prosthetic haem group containing an Fe2+ can bind to 4 molecules of O2 since it has 4 haem prosthetic groups Explain the role and structure of myoglobin Myoglobin is a monomeric protein with a globular structure. eight alpha helices connected by short non-helical regions. Topic 12 - Protein Function, Regulation + Oxygen Transport 1  Each myoglobin molecule contains one haem group with an iron ion (Fe2+) that binds with oxygen, (oxymyoglobin + deoxymyoglobin) Found in muscle tissues → oxygen storage molecule. Myoglobin has a high affinity for oxygen, producing a hyperbolic curve on a graph. Oxygen release from myoglobin helps muscle cells generate energy during anaerobic conditions. How does oxygen bind to haem groups - explain the structure of Haem and the desired charge of Iron Haem consists of a protoporphyrin ring and an iron (Fe) atom bound to nitrogen (N) atoms of the ring Iron in the +2 oxidation state is crucial for oxygen binding. MET - Metmyoglobin (myoglobin) and methaemoglobin (haemoglobin) are the +3 iron forms - Oxidizing iron to +3 destroys oxygen binding capacity. Topic 12 - Protein Function, Regulation + Oxygen Transport 2  Why is the oxygen binding curve for myoglobin hyperbolic? highly affinity → one haem group - binds to only one molecule of oxygen Myoglobin is fully saturated at a lower partial pressure of oxygen than haemoglobin Haemoglobin is giving up oxygen at a lower partial pressure, one that is equivalent to act of in tissue, whereas myoglobin is still holding onto it, makes it better for storage curve is more to the left - higher affinity Why is the oxygen binding curve for haemoglobin sigmoidal? Haemoglobin exhibits a sigmoidal oxygen-binding curve due to increasing binding affinity with more oxygen. Deoxyhemoglobin can exist in a T state or R state. Topic 12 - Protein Function, Regulation + Oxygen Transport 3  The T state has poor access to the haem group, making it less capable of accepting new oxygen. Oxygen binding promotes the stabilization of the R state through allosteric modification. Conformational changes in one subunit trigger changes in neighboring subunits. The first oxygen binding is challenging (low affinity), but subsequent oxygen binding becomes progressively easier. In the lungs (high pO2), haemoglobin is near 100% saturated due to its high affinity for oxygen. As oxygen partial pressure decreases, haemoglobin unloads oxygen because its affinity decreases, located near tissues for aerobic respiration What is an allosteric effector? Give some examples The ability of haemoglobin to reversibly bind O2 is affected by the pO2, pH of the environment, the partial pressure of carbon dioxide (pCO2), and the availability of 2,3-bisphosphoglycerate (2,3-BPG). 1. The cooperative binding of O2 allows haemoglobin to deliver more O2 to the tissues in response to relatively small changes in the pO2 (T, R State) 2. Low pH = low affinity for oxygen, High pH = high affinity for oxygen What change can allosteric activators/inhibitors cause to Haemoglobin specifically Haemoglobin's oxygen-binding is regulated by allosteric effectors. Topic 12 - Protein Function, Regulation + Oxygen Transport 4  The T conformation is the low-oxygen-affinity form The R conformation is the high-oxygen-affinity form activators shift curve to the left and enhance high affinity (R state) Allosteric inhibitors shift the curve to the left and enhance the low affinity (T state) Explain the Bohr Effect (CO2 and H+) Haemoglobin's oxygen binding affinity is inversely relate to acidity and Co2 conc. Therefore, release of O2 occurs: 1. where CO2 concentration is high (requires more O2 for respiration) 2. low pH - The Bohr effect reflects the fact that the deoxy form of haemoglobin has a greater affinity for H+ than does oxyhaemoglobin H+ and CO2 can both bind to haemoglobin molecules. -Binding of H+ and CO2 lowers the affinity of haemoglobin for oxygen - The Bohr Effect Metabolically active tissues produce large amounts of H+ and CO2 This affects the pH of the tissue Low pH = low affinity, High pH = high affinity The Bohr effect will shift the oxygen disassociation curve to the right hence increasing the P50 of haemoglobin therefore reducing the affinity of haemoglobin for oxygen. more to the right - more it dissociates Topic 12 - Protein Function, Regulation + Oxygen Transport 5  What is the effects on regulation of Oxygen binding with 2,3- bisphosphoglycerate Intermediate of Glycolysis One 2,3-BPG molecule binds per haemoglobin tetramer and decreases the affinity for O2 → curve is more to the right → more pO2 needed to saturate haemoglobin Allosterically inhibits O2 binding via T-state stabilisation BPG concentration increases at high altitudes, promoting O2 release at the tissues, Athletes train at high altitudes to train the body to release more oxygen to muscles if no 2,3-BPS was present in erythrocytes, the affinity of haemoglobin is almost that of myoglobin. why is this bad? 2,3-BPG is important in the unloading of oxygen to the tissues where it is needed so if it were absent, Hb would have a much higher affinity of oxygen Topic 12 - Protein Function, Regulation + Oxygen Transport 6  and it would be harder for O2 to dissociate Why is 2,3-BPG concentration important at high altitudes? Raised at higher altitudes so O2 is released more readily to the tissues as at higher altitudes where there's lower po2 Foetal Haemoglobin - Oxygen Binding HbF is a tetramer consisting of two α chains identical to those found in HbA, plus two γ gamma chains (α2γ2) HbF is the major haemoglobin in foetal blood. Higher binding affinity for O2 than HbA which allows transfer of oxygen to foetal blood supply from the mother. (graph more left) Under physiologic conditions, HbF has a higher oxygen affinity than does HbA. HbF is weakly binding to 2,3-BPG – This increases oxygen availability to the foetus What is β-thalassaemias genetic disorders where there is an imbalance between the number of alpha + beta globin chains Beta thalassaemias are a decreased/absent B-globin chain production, Alpha chains cant form stable tetramers, four subunit structure not produced Symptoms appear after birth → Inability or reduced ability to carry oxygen Topic 12 - Protein Function, Regulation + Oxygen Transport 7  What is α-Thalassaemias Alpha thalassaemias are a decreased or absent a-globin chain production β-chains can form unstable αβ dimers and therefore unstable tetramers with increased affinity for oxygen Symptoms occur before birth Explain the effects of carbon monoxide poisoning Carbon monoxide (CO): poison → combines with myoglobin and haemoglobin and blocks oxygen transport. Binds to haemoglobin 250x more readily than oxygen. When CO binds to one or more of the four haem sites, haemoglobin shifts to the R conformation, causing the remaining haem sites to bind O2 with high affinity, making it less likely to release oxygen. A patient suffering from carbon monoxide poisoning will experience symptoms such as dizziness, tiredness and unconsciousness due to oxygen deprivation. How can Carbon Monoxide poisoning be treated? Hyperbaric oxygen (HBO) therapy at 3x atmospheric pressure. Reduces the half-life of CO-haemoglobin (CO-Hb) Facilitates CO dissociation from haemoglobin. HBO allows oxygen (O2) to directly diffuse to tissues, bypassing the normal circulation. maintains tissue oxygenation while circulation recovers Lecture 12.2 - Regulation of Protein Function (Page 39 - CBG Notes) What are Protein Domains Topic 12 - Protein Function, Regulation + Oxygen Transport 8  region of a protein's polypeptide chain that is self-stabilizing and that folds independently from the rest Protein Regulation by Localization Cells control protein expression and activity but not protein location. Proteins can be specific or non-specific in their actions. Control of protein function involves managing when and where it operates. How is time control achieved in protein regulation Time control achieved by regulating transcription and degradation. 3 ways of protein localisation Localization methods include: Specific sequences guiding proteins to certain cellular regions. Post-translational modifications triggered by signal pathways. Binding to scaffold proteins that relay signals, they bring proteins together Protein Regulation by pH (Cathepsins) Cathepsins are lysosomal proteases. They need pH 5.5 to be active inactive at pH 7.4. An alpha helix initially blocks their active site. pH change alters the protein's conformation due to increased hydrogen ions, freeing the active site for normal function. → participating in the degradation of cellular components, Protein Regulation by pH (Diphtheria Toxin) Comprises A (catalytic) and B (regulatory) domains B domain binds to a receptor for cell entry Lysosomes, with their acidic environment, help break disulfide bonds between A and B domains Topic 12 - Protein Function, Regulation + Oxygen Transport 9  pH change causes the T domain to flip inside out and fuse with the cell membrane A domain enters the cytoplasm and binds to EF-2, blocking translation and causing cell death. GTP Binding Proteins Explain how Rho kinase is an example of GTP Binding Proteins Regulation by GAP (GTPase activating protein) and GEF (guanine nucleotide exchange factors): Ras activity is regulated by two proteins: a GEF (guanine nucleotide exchange factor) and a GAP (GTPase-activating protein). Topic 12 - Protein Function, Regulation + Oxygen Transport 10  GEF stimulates Ras to take up GTP, leading to its activation, initiating a signaling pathway. GAP stimulates Ras to hydrolyze GTP, causing it to be inactive. The activities of GEF and GAP are regulated in a coordinated manner to ensure proper control of Ras activity. EF-Tu - A Bacterial Translation Elongation Factor, how does it work? Works in elongation during translation Carries tRNA to the ribosome’s A site, can regulate translation by inhibiting or promoting the transport to the ribosome Can only bind to tRNA when it is bound to GTP GTP -> GDP hydrolysis results in release of tRNA Protein Movement (Myosin and actin) Myosin filaments have a head that is regulated by ATP activity Binding of ATP and subsequent hydrolysis of ATP to ADP results in the pushing of the myosin head along the actin Topic 12 - Protein Function, Regulation + Oxygen Transport 11  Membrane Bound Transporters - How does the Eukaryotic ABC Transporter work Control movement of molecules in and out of the cell. Molecules bind to receptors, can't escape. ATP binding induces a conformational change, pushing molecules out of the cell. How does CFTR Receptors work and how does Cystic Fibrosis Affect this CFTR receptor: Normally closed; chloride can't move out. Phosphorylation of the regulatory domain at a specific site opens the channel. ATP binding to secondary domains further opens the channel. In cystic fibrosis, if the CFTR channel can't open, chloride and water can't exit, leading to thick mucus, bacterial growth, lung infections, and potential death. Topic 12 - Protein Function, Regulation + Oxygen Transport 12  A comparison between Phosphorylated and GTP-binding Protein GEF (guanine nucleotide exchange factor) GAP (GTPase-activating protein). Muscle Glycogen Phosphorylase - how is it activated AMP and phosphate binding make it active. Activation is mediated by Protein Kinase A. cAMP (cyclic AMP) binds to Protein Kinase A. This binding causes the regulatory + catalytic subunits of the kinase to separate. Separation signals phosphorylation to happen, activating Muscle Glycogen Phosphorylase. Topic 12 - Protein Function, Regulation + Oxygen Transport 13  What is the role of Calmodulin modulator proteins and what is the role of calcium ions Calmodulin Modulator proteins bind to other proteins and cause conformational changes. They act as allosteric effectors that can activate or inhibit the target protein or enzyme. Calcium ions, for example, bind to calmodulin, forming a coordination sphere involving multiple amino acid residues simultaneously. Protein Regulation by Phosphorylation Topic 12 - Protein Function, Regulation + Oxygen Transport 14  how does Protein degradation work the proteasome is a critical component of the cellular machinery responsible for protein degradation. It ensures the removal of unwanted or damaged proteins, maintaining cellular homeostasis and playing a vital role in various biological processes. Protein Ubiquitination Topic 12 - Protein Function, Regulation + Oxygen Transport 15  Proteins that need to be degraded are typically tagged with a small protein called ubiquitin. This tagging process, known as ubiquitination, marks the target protein for destruction. What is Protein Glycosylation Glycosylation: the post-translational covalent addition of sugar molecules to asparagine, serine or threonine residues on a protein molecule. Glycosylation can add a single sugar or a chain of sugars at any given site. What is Protein lipidation Topic 12 - Protein Function, Regulation + Oxygen Transport 16  What is the role of ubiquitin Small proteins covalently attached to influence protein lifetime Topic 12 - Protein Function, Regulation + Oxygen Transport 17