Chapter 5: Protein Function PDF

Chapter 5: Protein Function -Myoglobin and Hemoglobin- Objectives: Examine the roles and mechanisms of protein-ligand interactions. Assess the oxygen binding properties of myoglobin and hemoglobin. Interpret the mechanisms of physiological regulation of oxygen deliv...

Chapter 5: Protein Function -Myoglobin and Hemoglobin- Objectives: Examine the roles and mechanisms of protein-ligand interactions. Assess the oxygen binding properties of myoglobin and hemoglobin. Interpret the mechanisms of physiological regulation of oxygen delivery. Identify the mechanisms of the allosteric regulation of hemoglobin. Summarize the molecular basis of sickle cell anemia and how it relates to malaria. Text Readings: Stryer 2nd Edition All of Chapter 9  Protein Function -Static vs Dynamic- Thus far, the proteins studied have been static in their functions. In this chapter will introduce a protein with dynamic functions. Some proteins have structurally flexibility and are capable of changes in conformation that relate to dynamic physiological functions. These changes to structure and function are often influenced by interaction with other molecules. Hemoglobin is a useful model to understand dynamic protein structure and function.  Protein Function -Ligands- Many proteins undergo reversible interactions with other molecules. These interactions can serve to regulate protein function. A molecule reversibly bound by the protein is called a ligand. A ligand can be any kind of molecule, including another protein. Protein-Ligand Interaction  Protein Ligands -Specificity- A ligand binds at a specific site on the protein called the binding site. The binding site is usually complimentary to the ligand in terms of shape, charge, hydrophobicity, hydrogen bonding potential A given protein may have multiple binding sites for multiple ligands. For example, ligands of hemoglobin include oxygen and oxygen and 2,3 2,3 bisphosphoglycerate bisphosphoglycerate (2,3 (2,3 BPG). BPG).  Protein Ligands -Induced Fit- The binding of a ligand may cause a conformational change in the protein. This induced fit can change the properties of the protein. These changes in protein structure often relate to changes in function. https://en.wikipedia.org/wiki/Enzyme_catalysis#/media/File:Hexo kinase_induced_fit.svg  Oxygen Delivery and Storage -Overview- The Challenge: Every cell requires a constant supply of oxygen. "BIOLOGICAL size of PROBLEM" determines the organisms. The Obstacles: For many multi-cellular organisms, the solubility of oxygen is too low to meet oxygen requirements through passive diffusion. Amino acid side chains not well suited for reversible binding of oxygen. Transition state metals have strong tendency to bind oxygen but produce damaging free radicals. The Solution: Specialized proteins for oxygen storage and delivery. Heme groups to safely harness iron’s oxygen binding properties.  Oxygen Delivery and Storage Systems -Overview- Myoglobin and hemoglobin serve distinct, but complimentary, physiological roles. Myoglobin and hemoglobin share many structural and functional features. Myoglobin (Mb): monomeric protein that facilitates oxygen storage in peripheral tissue. = quaternary 4 02. Hemoglobin (Hb): tetrameric protein found in can bind low affinity red blood cells that transports oxygen from lungs to the periphery. more high affinity http://www.phartoonz.com  Oxygen as a Limiting Resource Oxygen is poorly soluble in aqueous solutions. Emergence of larger, multi-cellular organisms depended on the evolution of proteins that could transport and store oxygen. The amount of available oxygen which can be delivered within the organism can limit it’s size. Insects grown in the presence of elevated oxygen can achieve greater sizes.  Reversible Oxygen Binding -Heme Prosthetic Groups- Cellular iron is bound in forms that sequester it and/or make it less reactive. Heme consists of a protoporphyrin ring system bound to a single (Fe2+) iron atom. Fe2+ binds O2 reversibly, Fe3+ does not bind O2. NO REDOX RYNS !! -> 4 ring system. O The ring system provides four coordinating interactions with the iron atom. The electron-donating characteristic of nitrogen prevent the conversion of Fe2+ to Fe3+. Myoglobin and hemoglobin both use heme. Heme is bound with discrete pockets of myoglobin and hemoglobin.  Reversible Oxygen Binding -Heme Prosthetic Groups- Fe2+ seeks six coordinating interactions: four come from interactions with heme. 4 NITROGENS wIn HEME a fifth comes interaction with an imidazole group of a proximal histidine residue. the sixth position is for O2 binding. A distal histidine provides a stabilizing interaction for bound O2. Distal His (closer Proximal His  structure Heme Prosthetic Groups -Carbon Monoxide Poisoning- function Carbon monoxide (CO) has a similar molecular structure as oxygen (O2). O2 CO Carbon monoxide exerts its deadly effects by competing with oxygen for binding to heme. CO binds heme with 200 times greater affinity than O2. https://en.wikipedia.org/wiki/Carbon_monoxide_p oisoning#/media/File:CO_toxicity_symptoms_(en ).jpg  Oxygen Binding Proteins -Myoglobin vs Hemoglobin (Structures)- IRON , HEME PROTEIN , Myoglobin, with a single sub-unit, is an example of tertiary structure. With a single heme group, myoglobin can bind one oxygen molecule. Hemoglobin, with four sub-units, is an example of quaternary structure. With four heme groups, hemoglobin can bind four oxygen molecules. Each sub-unit of hemoglobin closely resembles myoglobin. CO 4 MYOGLOBIN = 1 HEMOGLOBIN O2  Oxygen Binding Proteins -Myoglobin vs Hemoglobin (Functions)- Myoglobin has a higher affinity for oxygen than hemoglobin. Myoglobin has a hyperbolic curve of oxygen binding. Binding of oxygen by hemoglobin displays sigmoidal behavior. This indicates coopertivity of oxygen binding. CO myoglobinfinite than in O2 partial pressure of oxygen.  Myoglobin -Structure- Myoglobin is a small globular protein consisting of: a single polypeptide of 153 residues arranged in eight α-helices. a heme (iron porphyrin) prosthetic group. Sperm whale myoglobin was first protein structure determined by x-ray crystallography. ↳ whales oxygen = have to carry 02 for long time LOTS Of myoglobin. John Kendrew with model of myoglobin in progress. Copyright by the Laboratory of Molecular Biology in Cambridge, England.  Myoglobin 100 four of O2 in air. -Oxygen-Saturation Curve- The oxygen saturation curve of myoglobin is hyperbolic, indicating a single O2 binding constant. The amount of O2 required to half saturate the protein is quantified by P50. The P50 of myoglobin is 3 torr. > only binds a single 02 - To put this in a physiological perspective, the pO2 in the lungs (where physiological O2 concentrations are highest) is typically 100 torr and 20 torr in the periphery (where O2 concentrations are lowest). ↳ not a single binding constant > - quaternary structure % saturated 100  Myoglobin -Fraction Saturation- The fraction of myoglobin saturated with oxygen at a given partial pressure of oxygen is calculated by: X saturation θ= [pO2] / ([pO2] + [P50]) ↑ partial pressure artial pressure of Oxygen " at 50. In peripheral tissues, the partial pressure of O2 is around 20 torr. θ = 20/(20 + 3) = 87% saturation In the lungs, the partial pressure of O2 is 100 torr. θ = 100/(100 + 3) = 97% saturation. Myoglobin has a very high affinity for oxygen and is normally nearly saturated with oxygen everywhere in the body.  Oxygen Transport in the Blood -Hemoglobin- Hemoglobin contained within erythrocytes (red blood cells). Hemoglobin is an example of quaternary structure (α2β2) with four sub-units, each of which can bind an oxygen molecule. Hemoglobin is an allosteric protein whose oxygen affinity is regulated · through various physiological signals. can carry 402 molecules. can 2 adopt different , conformations 02 ↳ in differ affinity. T STATE = saturated wh R STATE "inactive" oxygen. "active"  Allosteric Proteins -General- The word allosteric is derived from the Greek words allos (meaning other) and stereos (meaning structure). Allosteric = Other Structure Allosteric proteins have T (inactive) and R (active) forms. transitioning These T and R forms are in rapid equilibrium. regulate overall affinity for oxygen. T R  (carry) Allosteric Proteins high affinity > - lungs -Hemoglobin- low affinity - > periphery (delivers A protein that binds O2 with high and constant affinity would saturate effectively with O2 in the lungs but not release it to tissues. R STATE A protein with a lower O2 affinity would be able to release O2 to tissues would not have sufficient affinity to saturate in the lungs. Hemoglobin solves the problem by undergoing transitions from high and low affinity states. Proteins with a single ligand-binding site (like myoglobin) cannot achieve this cooperative effect. R STATE - doesn't deliver O2. carries it along T STATE -wouldn'tfullysaturataearly  Allosteric Proteins -Modulators (Effectors)- ai distinc t from ding i o nal A bin gite can influence equilibrium M of proteins. Allosteric effectors (modulators) bind allosteric proteins at specific sites. Allosteric effectors can be either activators or inhibitors. active inactive Allosteric activators stabilize the R state; allosteric inhibitors stabilize the T state. When the normal ligand and modulator are the same, the interaction is homotropic. homo-same When the modulator is different from the normal ligand the interaction is heterotropic. hetero-different  Allosteric Properties of Hemoglobin a adopt quaternary > - specifictractions conformation specific > - The binding and release of O2 from Hb are allosterically regulated. For example, O2 is a homotropic allosteric activator of hemoglobin. Binding of the first O2 by hemoglobin causes a conformational change making easier to bind subsequent O2 (positive cooperativity). O2 binding promotes and stabilizes the R state of hemoglobin which has higher O2 affinity than the T state. Post - bindoseechle sensedodedate COOPERATIVITY a absence x = all RRRR TT O2 TT O2 RT O2 RR O2 O2 RR ↑ TT TT TT RR RR RT O2 O2 TT q conformational all saturate ↑ change FIT wi Oxygen TT = INDUCED TT  Allosteric Properties of Hemoglobin -T to R Transition- With T state hemoglobin, the iron atom is just outside the plane of the heme ring. With transition to the R state (O2 bound) the iron moves into plane of the ring. This minor movement within one subunit causes structural changes that are translated to the quaternary structure of the protein. fied extremely Briplicatenary subtle at astructure > - change  release y of O2 T Hemoglobin load when torr -Oxygen-Saturation Curve- matches ↓ max. 50 % Of capacity Critical to look at changes in the saturation of hemoglobin over the physiological range of O2. At the partial pressures of oxygen found in the ↑ lungs, Hb completely saturates with O2. sensitive At the partial pressures of oxygen found in the to P P of. periphery, Hb releases over half of its O2 load. changes The P50 of hemoglobin closely matches the 02 - partial pressures of O2 found in periphery. Hb is most sensitive for O2 release at the partial pressures of O2 found in the periphery. This allows Hb to sense and respond to changes in O2 levels in regions at greatest risk for hypoxia.  Allosteric Inhibition of Hemoglobin: 2,3 Bisphospho-D-glycerate resembla Initial investigations with highly purified missing 2 , 3-bisphospho- givcerate- h hemoglobin indicated an extremely high affinity for oxygen. This would limit the ability of the protein to release oxygen to the periphery. Replacing various components of blood revealed that 2,3 bisphosphoglycerate decreased hemoglobins’ affinity for oxygen. inhibitor to decreaseO2 affinity in hemoglobin. 2,3 Bisphosphoglycerate is a heterotropic allosteric inhibitor of hemoglobin.  Allosteric Inhibition of Hemoglobin: 2,3 Bisphospho-D-glycerate promote and stabilizeT state > - - inhibitor (affinity) netero - 2,3 BPG carries five units of negative charge. The pocket formed at the interface between the subunits of deoxyhemoglobin contains six positively charged residues. This pocket is unique to deoxyhemoglobin. ·  Fetal Hemoglobin -2,3 Bisphospho-D-glycerate- pulls O2 from maternal blood - higher affinity is needed to gain A fetus “breathes” in the womb by stripping 02 - O2 away from the maternal blood. Fetal Hb has a higher oxygen affinity than adult Hb. Adult Hb has six (+) residues at the 2,3BPG binding site, fetal Hb has four. 2 different genes present – His143 replaced by Ser Decreased affinity for 2,3 BPG translates into higher O2 affinity for fetal Hb. Lower affinity for the allosteric inhibitor bestows higher affinity for O2. less o charge I amino affinity for = lower acid > - inhibitor difference ↳ = higher 02 affinity.  less 02 - > decrease affinity of inhibitors. BPG ↓ Sufficient O2 delivery -High Altitude Adaptation-. Slightly less O2 in lungs. There is less O2 at high altitude. Adaptation to high altitude can rapidly occur through increased production of 2,3 BPG. Increased 2,3 BPG decreases Hb’s O2 affinity to ensure sufficient O2 delivery to the periphery. Activity at extreme altitudes usually requires artificial means to provide O2. Breckenridge Colorado (altitude 10,000 feet)  PH dependence The Bohr Effect of hemoglobins affinity for Oxygen- The Bohr Effect describes the pH dependence of hemoglobin s affinity of O2. At decreased pH hemoglobin has a lower affinity for O2. Active tissues have lower pH due to: decrease affinity increased muscle activity increases release production of CO2. As we will see, this more CO2 eventually decreases pH. oxygen. In extreme exercise, muscles produce lactic acid to further decrease pH. The Bohr effect serves to coordinate increased release of oxygen to active tissues.  Coordination of O2 Delivery and CO2 Removal There are two primary challenges to cellular respiration and metabolism: 1) Delivering sufficient O2 to the tissues. 2) Removing CO2 (the exhaust of metabolism) from the periphery. Important to consider that both the oxygen requirements and carbon dioxide production increase with increased muscle activity. The body has adapted effective strategies to coordinate these needs. oxygen required to fuel process output of CO2. > -  Coordination of O2 Delivery and CO2 Removal -Mechanism #1- of ~ location CO2 is taken up into red blood cells and hemoglobin. converted to bicarbonate and a proton by the enzyme carbonic anhydrase. rect CO2 Wl120 > - bicarbonate CO3. Through this reaction: 1) CO2 is converted into a soluble form for transport to the lungs. o acidic pH more 02 = 2) The decreased pH decreases release hemoglobin s O2 affinity to promote O2 release to active tissues. The more active the tissues, the greater the production of CO2, the greater the production of CO2 the greater the release of O2.  Coordination of O2 Delivery and CO2 Removal -Mechanism #2- more acidic lower affinity = CO2 can form a covalent carbamate linkage to the N terminus of each chain of hemoglobin chain to form carbaminohemoglobin. This reaction has three important outcomes: Converts CO2 into a more soluble form to assist in its transport to the lungs. Carbamino hemoglobin has a lower O2 affinity than hemoglobin to promote O2 release. decrease pH The released proton promotes O2 release allows for increase through the Bohr Effect. oxygen released to tissues.  Sickle Cell Anemia is a Molecular Disease of Hemoglobin circulatory disease - > not enough Q to get Of Results from a single amino acid change to periphery (Glu6Val). body. Formation of fibers from the deoxy forms of HbS. Fibers tend to form in the capillaries (where the O2 concentration is the lowest) which blocks blood flow to the extremities of the body. t state = peripheral of body. ↓ hydrophobic pocket Healthy Sickle Cell Anemia = Glueval valine = non-polar , doesn't want De-oxygenated Oxygenated De-oxygenated Oxygenated TO be exposed to M20.  Sickle Cell Anemia -Malaria- SCA primarily affects African Americans and Africans; selected for in regions where malaria imposes a selection pressure. One theory of why individuals heterozygous for SCA have resistance to malaria. Malaria infects red blood cells. Infection decreases pH in red blood cells. Decreased pH causes release of oxygen from Hb. For individuals with sickle cell anemia, deoxy HbS forms fibers deforming the red blood cell. These deformed red blood cells (which contain malaria) are Electron micrograph of a Plasmodium selectively destroyed by the spleen. falciparum-infected red blood cell (center).  Other Oxygen Transport Proteins -Hemocyanin- Some invertebrates, like horseshoe crabs, use hemocyanin, rather than hemoglobin, to carry oxygen. As an oxygen transporter, hemocyanin is distinct from hemoglobin in that: Hemocyanin uses copper rather than iron (blue blood rather than red). Two copper atoms bind a single oxygen molecule. There is no heme ring group, the copper atom is coordinated through histidine residues. Hemocyanin is not localized within specialized oxygen-transport cells.  Copyright Sourcing Images/Figures/Tables from Textbook – Permission: Courtesy of MacMillan Learning. Slide 3: Source: https://en.wikipedia.org/wiki/Docking_(molecular)#/media/File:Docking_representation_2.png Permission: CC BY-SA 4.0 Courtesy of Scigenis. 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Permission: This material has been reproduced in accordance with the University of Saskatchewan Fair Dealing Guidelines, an interpretation of Sec.29.4 of the Copyright Act. Slide 14: Source: https://www2.mrc-lmb.cam.ac.uk/photo-archive/john-kendrew-with-model/ Permission: Courtesy of the Medical Research Council.  Copyright Sourcing Slide 19: Source: Lehninger Principles of Biochemistry (2008) 5th Edition, page 161. Permission: This material has been reproduced in accordance with the University of Saskatchewan Fair Dealing Guidelines, an interpretation of Sec.29.4 of the Copyright Act. Slide 33: Source: https://en.wikipedia.org/wiki/Malaria#/media/File:Red_blood_cells_infected_with_malaria.jpg Permission: CC BY 2.0 Courtesy of Rich Fairhurst and Jordan Zuspann.