Globular Proteins PDF

GLOBULAR PROTEINS DR IMAKANDO M  OVERVIEW  Introduction  Myoglobin  Introduction  Structure and function  Oxygen Binding and Oxygen Dissociation curve  Related diseases  Hemoglobin  Introduction  Types of hemoglobin...

GLOBULAR PROTEINS DR IMAKANDO M  OVERVIEW  Introduction  Myoglobin  Introduction  Structure and function  Oxygen Binding and Oxygen Dissociation curve  Related diseases  Hemoglobin  Introduction  Types of hemoglobin  Functions and Normal levels  Oxygen dissociation Curve and factors affecting it  Heme group  Similarities and differences  INTRODUCTION  Amino acid chains fold into shapes that resemble spheres and are called globular proteins  This type of folding increases solubility of proteins in water  Polar groups on the protein’s surface  Hydrophobic groups in the interior  Fibrous proteins are mainly insoluble structural proteins  Hemeproteins are a group of specialized proteins that contain heme as a tightly bound prosthetic group  Hemoglobin: oxygen transport function  Myoglobin: oxygen storage/supply function in heart and muscle  INTRODUCTION CONTIN…..  Myoglobin and hemoglobin are hemeproteins whose physiological importance is principally related to their ability to bind molecular oxygen.  Hemoglobin is a heterotetrameric oxygen transport protein found in red blood cells (erythrocytes),  Whereas myoglobin is a monomeric protein found mainly in muscle tissue where it serves as an intracellular storage site for oxygen.  The oxygen carried by hemeproteins such as hemoglobin and myoglobin is bound directly to the ferrous iron (Fe2+) atom of the heme prosthetic group.  Oxidation of the iron to the ferric (Fe3+) state renders the molecule incapable of normal oxygen binding.  When the iron in heme is in the ferric state, the molecule is referred to as hemin.  MYOGLOBIN  It is a monomeric protein found in the muscles  Myoglobin is an example of a globular and tertiary structure protein.  A large, coiled polypeptide called globin makes up most of the molecule  Used in storage and transportation of oxygen within the muscle.  Binds a single oxygen molecule and it carries O2 from capillaries to sites of usage in cells.  It contains Heme group  STRUCTURE  The functional unit of myoglobin is an iron—porphyrin complex that is embedded in the protein.  Myoglobin has eight α -helical regions (resembles subunits of hemoglobin).  Starting at the N-terminal, these are termed helices A – H.  75% of the residues are present in the 8 right handed α helices.  There about 7 – 20 amino acid residues in these α helices.  Hydrogen bonding in the polypeptide backbone stabilizes the α helical regions; amino acid side chains are also involved in hydrogen bonds.  The complete polypeptide of myoglobin is made up of 153 amino acids.  Two polar histidine residues are found in the interior: they interact with the heme and bound oxygen and thus play a role in function of the protein. STRUCTURE OF MYOGLOBIN The charged amino acids are located on the surface The heme group is present at the center of the molecule Heme is a large, aromatic porphyrin ring with four pyrrole nitrogen bound to a ferrous (Fe(II)) ion at the center Myoglobin gives red color to skeletal muscles The iron (Fe)-containing heme group allows myoglobin to reversibly bind to O2  If oxygen bonds to heme only and not on the polypeptide chain then what is the function of the polypeptide in myoglobin and heme? THREE MAIN REASONS:  The presence of a polypeptide prevents spontaneous oxidation of ferrous iron to ferric iron which cannot bind oxygen.  The presence of the polypeptide chain makes oxygen binding reversible and  Significantly decreases carbon monoxide affinity for myoglobin (and hemoglobin)  02 BINDING TO MYOGLOBIN  Myoglobin coordinates O2 reversibly and controls its concentration in tissue.  Deoxymyoglobin (Mb) is bluish red and contains Fe(II); this is the oxidation state that binds 02 to give the familiar bright red oxymyoglobin (oxyMb).  The Fe in deoxymyoglobin is five-coordinate, high-spin, and lies above the plane of the ring.  When 02 binds, it is coordinated end-on to the Fe atom, the electronic structure of which is tuned by the F helix histidine ligand.  The unbound end of the 02 molecule is fastened by a hydrogen bond to the imidazole-NH of the histidine in helix E.  In some instances deoxymyoglobin becomes oxidized to Fe(III), which is called metmyoglobin (metMb) and is unable to bind 02.  02 BINDING CURVE  The shape of the 02-binding curve of myoglobin can be described mathematically by the equation:  Myoglobin's Oxygen-Binding Curve Is Hyperbolic.  The curve for myoglobin can be described by a simple equilibrium between deoxy- and oxymyoglobin  In contrast, the 02-binding curve of hemoglobin is sigmoid (S) shaped but the S-shaped curve for hemoglobin can be described only in terms of a cooperative interaction between the four hemes OXYGEN-HEMOGLOBIN DISSOCIATION CURVE OXYGEN-HEMOGLOBIN DISSOCIATION CURVE  As shown in the previous slide, at low oxygen pressures, the affinity of deoxyhemoglobin for 02 is substantially lower than that of myoglobin, whereas at high 02 pressures the two proteins have comparable 02 affinities.  The physiological consequences of the unusual S-shaped 02-binding curve of hemoglobin are enormous.  In the lungs, where 02 pressure is highest, the high oxygen affinity of deoxyhemoglobin allows it to be completely loaded with 02, giving four 02 molecules per hemoglobin. OXYGEN-HEMOGLOBIN DISSOCIATION CURVE  In the tissues, however, where the oxygen pressure is much lower, the decreased oxygen affinity of hemoglobin allows it to release 02, resulting in a net transfer of oxygen to myoglobin.  The shape of myoglobin's oxygen binding curve is hyperbolic, meaning that it holds onto oxygen much tighter.  Only when the amount of oxygen is extremely low, as in the mitochondria of working muscle, does myoglobin release its oxygen.  There, the oxygen is used in cellular respiration to produce energy  MYOGLOBIN RELATED DISEASE Myoglobinuria:  Myoglobin is excreted in urine due to muscle damage (rhabdomyolysis)  May cause acute renal failure  Specific marker for muscle injury  Less specific marker for heart attack  HEMOGLOBIN  A major globular protein in humans  Composed of four polypeptide chains:  Two a and two b chains  Adult hemoglobin is a heterotetrameric [α(2):β(2)] hemeprotein found in erythrocytes.  Hemoglobin is abbreviated as Hb or Hgb.  Held together by non-covalent interactions  Each chain is a subunit with a heme group in the center that carries oxygen  A Hb molecule contains 4 heme groups and carries 4 molecules of O2  INTRODUCTION  The overall structure of hemoglobin is α₂β₂  Both the α- and β-chains of hemoglobin are very similar to the myoglobin chain  The α-chain is 141 residues long, and the β-chain is 146 residues long  Amino acids of α-chain and the β-chain are homologous(same amino acid residues are in the same positions).  The heme group of myoglobin and hemoglobin is the same  INTRODUCTION CONTIN……  Found in red blood cells.  Carries oxygen to tissues and transport carbon dioxide and hydrogen ions back to lungs.  Binds to a total of 4 oxygen molecules  Both hemoglobin and myoglobin bind oxygen reversibly but hemoglobin exhibits positive cooperativity.  This means that binding of one Oxygen molecule, makes it easier for the other Oxygen molecules to bind.  This binding can be shown in an oxygen binding graph for both myoglobin and hemoglobin  TYPES OF HEMOGLOBIN  Based on the non-alpha subunits, normal hemoglobin is mainly of three types:  Haemoglobin A  It is the predominant type of hemoglobin accounting for about 95 to 98% of total adult hemoglobin. It contains two alpha subunits and two beta subunits.  Hemoglobin A2  It accounts for about 2 to 3% of total adult hemoglobin. It contains two alpha subunits and two delta subunits.  Hemoglobin F  is the hemoglobin of fetuses and newborns and it is present in scanty amounts, below 1%, in adults. It contains two alpha subunits and two gamma subunits.  Besides these major hemoglobin, there are other mutated forms also like hemoglobin E, hemoglobin S, and hemoglobin C.  FUNCTIONS OF HEMOGLOBIN  The primary function of hemoglobin is to carry oxygen and carbon dioxide gases. It also serves the secondary function of maintaining blood pH and buffering the blood.  Oxygen Transport  Fe+2 ion of a heme group can bind one O2 molecule; hence, a total of 4 O2 molecules can be carried by one Hgb.  Carbon dioxide Transport  Fe+2 ion of a heme group can bind one CO2 molecule; hence, a total of 4 CO2 molecules can be carried by one Hgb.  When CO2 is bound with Hgb, it is called carbaminohemoglobin. About 20 to 25% (on average 23%) of CO2 in blood is carried by carbaminohemoglobin.  FUNCTIONS OF HEMOGLOBIN  Other Gases/Ion Transport  Besides O2 and CO2, Hgb can also bind with other ligands like carbon monoxide (CO), nitric oxide (NO), sulfur monoxide (SO), nitrite ion (NO-2), sulfides (S-2), etc.  The affinity of Hgb with CO is more than 200 times the affinity of Hgb with O2.  When CO is bound with Hgb, it is called carboxyhemoglobin.  Regulation of Blood pH and Buffering Function  Hgb molecules can also bind to the hydrogen ions and maintain the pH of the blood.  NORMAL HEMOGLOBIN LEVEL  The amount of hemoglobin in blood is expressed in grams per deciliter (g/dl).  The amount of hemoglobin depends on the age, sex, and health status of an individual.  In general, the Hgb level in a human range from 12 to 18 g/dl.  A COMPARISON OF THE OXYGEN BINDING BEHAVIOR OF MYOGLOBIN AND HEMOGLOBIN  The oxygen binding curve for myoglobin is hyperbolic where as that of hemoglobin is sigmoid.  Myoglobin has a higher affinity for O2 than haemoglobin  Myoglobin contains only a single globin chain: its dissociation curve is a rectangular hyperbola.  In the tetrameric form of hemoglobin, the binding of oxygen is, a cooperative process.  The oxygenation of each chain causes structural changes which increase the affinity of the heme of the remaining chains for oxygen.  This positive cooperative binding and increasing oxygen affinity as oxygen loads is the cause of the sigmoid shape of the dissociation curve  The relationship between concentration, partial pressure of O2 (PO2) and quantity of O2 bound to hemoglobin or myoglobin is expressed as an O2 saturation curve.  The binding of oxygen to heme increases as the concentration of oxygen in the environment increases and decreases as the oxygen concentration decreases.  The graph of hemoglobin indicates that the binding of the first oxygen molecule facilitates binding of a second oxygen molecule, which facilitates the binding of a third, which in turn facilitates the binding of the fourth.  This is precisely what is meant by the term cooperative binding  Hemoglobin binding curve is still lower than that of myoglobin at any oxygen pressure.  This shows that myoglobin has a higher percentage of saturation than hemoglobin at my given pressure  HEME GROUP  An example of a prosthetic group.  It is made up of a metal iron (Fe2+) and an organic protoporphyrin ring  Porphyrin part consists of a cyclic tetrapyrrole consisting of four molecules of pyrrole linked by 4 α-methylene bridges,2 propionate groups and 2 vinyl groups.  One atom of ferrous iron resides at the center of the planar tetrapyrrole.  The conjugated double bonds absorbs visible light and colors heme deep red.  The ability of myoglobin or hemoglobin to bind oxygen depends upon this heme group (prosthetic group). HEME GROUP  Iron atom has 6 coordination positions  4 to nitrogens that are part of the flat tetrapyrrole ring and 2 perpendicular to the ring  Coordinated N atoms help prevent conversion of the heme iron to the ferric (Fe3+) state.  Iron in the Fe2+ state binds oxygen reversibly but not in the Fe3+ state.  The 𝒕 coordination site is occupied by a side chain nitrogen of His F8 (proximal histidine) residue and the 𝒕 site is the binding site for molecular oxygen.  These 2 sites lie perpendicular to the ring on opposite sides.  The other histidine in the interior of the molecule His E7 (distal histidine) on the same side as oxygen acts as a gate that opens and closes as oxygen enters the hydrophobic pocket to bind to the heme.  It sterically inhibits oxygen from binding perpendicularly to the heme plane  Isolated heme binds carbon monoxide (CO) 250 times more strongly than oxygen.  CO is present in small quantities in the atmosphere and arises in cells from the catabolism of heme. CO does not completely displace O2 from heme iron  The accepted explanation is that the apoproteins of myoglobin and hemoglobin create a hindered environment.  In myoglobin and hemoglobin the distal histidine sterically prevents the orientation of Fe, C and O perpendicular to the plane of heme.  Binding at a less favored angle reduces the strength of the heme-CO bond.  Great excess of O2 over CO normally present, dominates heme-O2 bond  In deoxyhemoglobin, the Fe atom lies out of the porphyrin plane by about 0.04nm or about 0.06 nm.  As the Fe atom moves, it drags histidine F8 and the helix F along with it.  These shifts are transmitted to the subunits where they trigger conformational re-adjustments that lead to the rupture of interchain salt links.  Deoxyhemoglobin resists oxygenation because the deoxy form is stabilized by specific hydrogen bonds and salt bridges.  In deoxyhemoglobin, with all of these interactions intact, the C- termini of the four subunits are restrained, and this conformational state is termed T (the tense or taut) form.  The binding of the first O2 molecule to deoxyHb shifts the heme iron towards the plane of the heme ring.  This motion is transmitted to the proximal histidine F8 and to the residues attached to it.  The shift in helix F upon oxygenation leads to rupture of salt bridges between the carboxyl terminal residues of all four subunits.  In oxyhemoglobin, C-termini almost has complete freedom of rotation, and the molecule is now in its R (relaxed) form.  Oxygen is accessible only to the heme groups of the α-chains when hemoglobin is in the T conformational state and the heme of β-chains in the T state is virtually inaccessible because of steric hindrance by valine residue in the E helix.  This hindrance disappears when the hemoglobin molecule undergoes transition to the R state.  The conformational changes significantly increase the affinity of the remaining unoxygenated hemes for O2.  The terms T and R also are used to refer to the low affinity and high- affinity conformations, respectively  FACTORS INFLUENCING OXYGEN BINDING  The binding of oxygen to hemoglobin is loose and reversible.  It is governed by the following factors:  Partial pressure of oxygen (PO2)  Partial pressure of carbon dioxide (PCO2).  pH (acidosis) – the effect of H⁺ is also known as the Bohr effect  Temperature  2,3- bisphosphoglycerate HOW THE OXYGEN SATURATION CURVE OF HEMOGLOBIN IS AFFECTED BY DIFFERENT FACTORS  NOTE  Right shift indicates a decrease in hemoglobin’s affinity for oxygen, facilitating oxygen release to tissues.  A leftward shift indicates an increased affinity of hemoglobin for oxygen, making it less likely to release oxygen  Protons, carbon dioxide, and the metabolite 2,3- bisphosphoglycerate (or 2,3 BPG), all affect the binding of O2 to haemoglobin.  Low pH favors T form, which has low oxygen affinity and releases oxygen to the tissues.  Thus, as the pH decreases, dissociation of O2 from hemoglobin is enhanced.  When a tissue's metabolic rate increases, its carbon dioxide production increases.CO2 affect O2 binding to Hb similar to that of H+, partly because it produces H+ when it dissolves in the blood:  HCO3- causes the pH of tissues to decrease, and so, promotes the dissociation of oxygen from hemoglobin to the tissue, allowing the tissue to obtain enough oxygen to meet its demands.  Effect of Temperature  Increase in metabolic activity increases temperature.  Increase in temperature decreases Hb affinity for O2 by weakening and denaturing the bond between O2 and Hb.  Decreasing affinity increases O2 unloading to the tissues.  Effect of pH  When O2 binds to Hb protons are released due to conformational changes in the Hb.  Hb + O2 HbO2 + H+  Active tissue produces protons.  Low pH, the H+ protonates terminal amino acids driving it into the T-state. This increases oxygen unloading to the tissues.  Effect of CO2  Increased metabolic activity increases CO2. Most of the CO2 content (80 – 90%) is transported as bicarbonate ions. The formation of a bicarbonate ion will release a proton into the plasma.  Lowering the pH.  Increased O2 unloading to tissues.  Effect of 2,3-bisphosphoglycerate (2,3-BPG)  Produced in red cells  Production increased under conditions of low O2 such as anaemia and high altitude.  In tissues 2,3-BPG decreases affinity of Hb for O2 enhancing O2 unloading HOW THE OXYGEN SATURATION CURVE OF HEMOGLOBIN IS AFFECTED BY DIFFERENT FACTORS  NOTE  Right shift indicates a decrease in hemoglobin’s affinity for oxygen, facilitating oxygen release to tissues.  A leftward shift indicates an increased affinity of hemoglobin for oxygen, making it less likely to release oxygen  FETAL HAEMOGLOBIN  Fetal Hb differs from adult Hb in that the β-chains are replaced by very similar, 146-residue subunits called γ- chains - fetal Hb is α2γ2.  2,3 BPG binds less effectively with the γ-chains of fetal Hb (Hb F).  Hence fetal Hb has higher affinity for oxygen.  This enables the foetus to extract oxygen from maternal circulation.  Fetal γ-chains have Ser instead of His at position 143 (H21), and thus lack two of the positive charges required for 2,3 BPG binding.  DISEASES RELATED TO HEMOGLOBIN  Sickle Cell Disease  It is the condition when the body produces abnormal hemoglobin, hemoglobin S, due to a mutation in the beta-globin gene HBB.  Thalassemia  It is an inherited disease characterized by a decrease in hemoglobin production by the body. It is due to the reduction or complete absence of one or more globin subunits.  Polycythemia  It is characterized by increased hemoglobin levels in the blood.  Methemoglobinemia  It is the condition characterized by a reduction in the ability of hemoglobin to carry oxygen due to a change of iron from the reduced Fe+2 (ferrous) states to the oxidized Fe+3 (ferric) states.  Hemoglobinuria  Presence of hemoglobin in urine.  Hereditary persistence of fetal hemoglobin (HPFH)  It is a benign condition characterized by the presence of fetal hemoglobin, hemoglobin F, in adults in abundant quantity  SIMILARITIES & DIFFERENCE  Myoglobin has only one subunit and hemoglobin has four subunits (2α and 2β subunits).  The tertiary structure of myoglobin subunit and the four hemoglobin subunits are similar.  Both have a heme prosthetic group which is made up of a protoporphyrin ring and iron ion.  The ability of myoglobin or hemoglobin to bind oxygen depends upon this heme group (prosthetic group)  The iron atom in the center can form 6 bonds: 4 with nitrogens from protoporphyrin and 2 on either side of plane.  Iron atom can be in ferrous (+2) or ferric (+3) state and only +2 state binds oxygen.  Whereas myoglobin stores and transports oxygen within muscle cells, hemoglobin binds and transports oxygen, protons, carbon dioxide in blood circulation.  In addition, the binding of oxygen to hemoglobin is cooperative and is regulated by pH, CO2 and 2,3-bisphosphoglycerate (BPG), which is not observed with binding of oxygen to myoglobin  Saturation of myoglobin is higher at all oxygen pressures than hemoglobin which means myoglobin has higher affinity for oxygen than does hemoglobin.  The oxygen dissociation curve of myoglobin is hyperbolic and that of hemoglobin is sigmoidal implying that the binding of oxygen to hemoglobin is cooperative. THANK YOU