血红蛋白和免疫球蛋白ppt课件

1、Concepts 基本概念 2、Reversible Binding of a Ligand to a Protein: 肌红蛋白和血红蛋白 3、Complementary Interactions between Proteins and Ligands: 免疫系统和免疫球蛋白 2.4 蛋白质的结构和功能 本次作业（第三次作业） 1.海拔高度调控别构效应子BPG浓度的分子 基础（或可以理解为海拔高度如何决定代 谢产物BPG的浓度）。 2.免疫记忆的分子基础。 配基(ligand): A molecule bound reversibly by a protein is called a ligand. A ligand may be any kind of molecule, including another protein. A ligand binds at a site on the protein called the binding site, which is complementary to the ligand in size, shape, charge, and hydrophobic or hydrophilic character. 1、Concepts 基本概念 The binding of a protein and ligand is often coupled to a conformational change in the protein that makes the binding site more complementary to the ligand, permitting tighter binding. The structural adaptation that occurs between protein and ligand is called induced fit (诱导契合). In a multisubunit protein, a conformational change in one subunit often affects the conformation of other subunits. Intermolecular signal transduction 结合常数 解离常数 低解离常数与亲和层析 Enzymes represent a special case of protein function. Enzymes bind and chemically transform other molecules- they catalyze reactions. The molecules acted upon by enzymes are called reaction substrates (底物) rather than ligands, and the substrate- binding site is called the catalytic site (催化位点) or active site (活性位点). 底物和活性位点 Interactions between ligands and proteins may be regulated, usually through specific interactions with one or more additional ligands. These other ligands may cause conformational changes in the protein that affect the binding of the first ligand. (for example, the case of BPG) Allosteric (变构效应) - an effect that affects the activity of one part of an enzyme (such as an active site) by the binding of a molecule at a different site (regulatory site) at a different location on the enzyme. 变构效应/别构效应 Changes in conformation may be subtle, reflecting molecular vibrations and small movements of amino acid residues throughout the protein. A protein flexing (挠动) in this way is sometimes said to “breathe” Grd19/SNX3 1 1 2 2 3 3 1 1 2 2 33 4 4 1 C N N C Grd19- PtdIn(3)P 蛋白质的柔性 (Proteins are flexible) Grd19/SNX3 1 33 PX domain 158 162 phosphatidylinositol-3-phosphate PtdIn(3)P 磷脂酰肌醇-3-磷酸 Kd=0.150.5 M Active Form Changes in conformation may also be quite dramatic, with major segments of the protein structure moving as much as several nanometers. Specific conformational changes are frequently essential to a proteins function. LicT mutant (active) H207D/H269D LicT wt (inactive) H207/H269 phosphorylation 2、Reversible Binding of a Ligand to a Protein: 肌红蛋白和血红蛋白 血红蛋白: hemoglobin-oxygen transport protein (22 in complex with 4 hemes) 肌红蛋白: myoglobin-oxygen storage protein Myoglobin and hemoglobin may be the most-studied and best-understood proteins. These molecules illustrate almost every aspect of that most central of biochemical processes: the reversible binding of a ligand to a protein. This classic model of protein function tells us a great deal about how proteins work. globin (珠蛋白) in complex with heme (血红素) In 1840, the oxygen-carrying protein haemoglobin was discovered by Hnefeld. In 1851, Otto Funke published a series of articles in which he described growing hemoglobin crystals by successively diluting red blood cells with a solvent such as pure water, alcohol or ether, followed by slow evaporation of the solvent from the resulting protein solution. In 1958, John Kendrew and associates successfully determined the structure of myoglobin by high-resolution X-ray crystallography. In 1959, Max Perutz determined the molecular structure of hemoglobin by X-ray crystallography. For this discovery, John Kendrew shared the 1962 Nobel Prize in chemistry with Max Perutz. 1) Kendrew, JC. Bodo, G. Dintzis, HM. Parrish, RG. Wyckoff, H. and Phillips DC. (1958). “A Three- Dimensional Model of the Myoglobin Molecule Obtained by X-Ray Analysis“. Nature 181 (4610): 662666. 2) Perutz, M.F.; Rossmann, M.G.; Cullis, A.F.; Muirhead, H.; Will, G.; North, A.C.T. (1960). “Structure of H“. Nature 185 (4711): 416422. 3) Perutz MF (November 1960). “Structure of hemoglobin“. Brookhaven symposia in biology 13: 16583. Research history 1) The sequences of hemoglobins differ between species. 2) Even within a species, different variants of hemoglobin exist. 3) Mutations in the genes for the hemoglobin protein in a species result in hemoglobin variants, some of these mutant forms of hemoglobin cause a group of hereditary diseases termed the hemoglobinopathies. 4) The best known is sickle-cell disease, which was the first human disease whose mechanism was understood at the molecular level. 5) A (mostly) separate set of diseases called thalassemias involves underproduction of normal and sometimes abnormal hemoglobins, through problems and mutations in globin gene regulation. 6) All these diseases produce anemia. Genetics Types in humans Hemoglobin variants are a part of the normal embryonic and fetal development, but may also be pathologic mutant forms of hemoglobin in a population, caused by variations in genetics. Some variants such as sickle-cell anemia are responsible for diseases (hemoglobinopathies). Other variants cause no detectable pathology (non- pathological variants). In the embryo: Gower 1 (22) Gower 2 (22) (PDB 1A9W) Hemoglobin Portland (22) In the fetus: Hemoglobin F (22) (PDB 1FDH) In adults: Hemoglobin A (22) (PDB 1BZ0) -The most common with a normal amount over 95% Hemoglobin A2 (22) - chain synthesis begins late in the third trimester and in adults, it has a normal range of 1.5-3.5% Hemoglobin F (22) - In adults Hemoglobin F is restricted to a limited population of red cells called F-cells. However, the level of Hb F can be elevated in persons with sickle-cell disease. Expression of human globin genes at different stages of development. 1) Hemoglobin (Hb) is synthesized in a complex series of steps. 2) The heme part is synthesized in a series of steps in the mitochondria (线粒体) and the cytosol of immature red blood cells, while the globin protein parts are synthesized by ribosomes in the cytosol. 3) Production of Hb continues in the cell throughout its early development from the proerythroblast (原成红细胞) to the reticulocyte (网织红细胞) in the bone marrow (骨髓). 4) The nucleus is lost in mammalian (哺乳动物) red blood cells, but not in birds and many other species. Even after the loss of the nucleus in mammals, residual ribosomal RNA allows further synthesis of Hb until the reticulocyte loses its RNA soon after entering the vasculature (脉管系统). Synthesis Role of the globins in oxygen transport and storage. hemoglobin myoglobin 静脉 动脉 肺/腮 The iron atom of heme (亚铁 血红素) has six coordination bonds: four in the plane of, and bonded to, the flat porphyrin ring system. Porphyrins (卟啉), of which protoporphyrin (原卟啉) IX is only one example, consist of four pyrrole (吡咯) rings linked by methene (亚甲基) bridges, with substitutions at one or more of the positions denoted X. Heme (亚铁血红素) This view shows the two coordination bonds to Fe2 perpendicular to the porphyrin (卟 啉) ring system. One of these two bonds is occupied by a His residue, sometimes called the proximal His. The other bond is the binding site for oxygen. The remaining four coordination bonds are in the plane of, and bonded to, the flat porphyrin ring system. The heme group viewed from the side. Two coordination bonds perpendicular (垂直于) to the plane. Evolution of the globin genes 圆口鱼类 多骨鱼类 灵长类 哺乳动物 Evolutionary conservation of the globin folding pattern The structure of myoglobin Myoglobin (a)Oxygen binds to heme with the O2 axis at an angle, a binding conformation readily accommodated by myoglobin. (b)Carbon monoxide binds to free heme with the CO axis perpendicular(垂直) to the plane of the porphyrin (卟啉) ring. When binding to the heme in myoglobin, CO is forced to adopt a slight angle because the perpendicular arrangement is sterically blocked by His E7, the distal His. This effect weakens the binding of CO to myoglobin. (c)Another view (derived from PDB ID 1MBO), showing the arrangement of key amino acid residues around the heme of myoglobin. The bound O2 is hydrogen-bonded to the distal His, His E7 (His64), further facilitating the binding of O2. Steric effects on the binding of ligands to the heme of myoglobin Dynamics of oxygen release by myoglobin The rate-limiting process in oxygen release is the opening of a pathway for the O2 molecule to escape from the heme pocket. Oxygen may spend time “rattling in its cage“ - and perhaps being recaptured - before the tertiary structure of the myoglobin shifts enough to let it escape 拨浪鼓 Dominant interactions between hemoglobin subunits. Hemoglobin A comparison of the structures of myoglobin (PDB ID 1MBO) and the subunit of hemoglobin (derived from PDB ID 1HGA). The looser conformation is called relaxed (松弛的) (R). The tighter conformation is called tense (紧张的) (T). The energy price for the change from the T state to the R state is paid by the binding of O2 to the molecule. Once the O2 has departed, the molecule will naturally fall back into its lower-energy deoxy conformation (T). 1) In the tetrameric form of normal adult hemoglobin, the binding of oxygen is a cooperative process. 2) The binding affinity of hemoglobin for oxygen is increased by the oxygen saturation of the molecule, with the first oxygens bound influencing the shape of the binding sites for the next oxygens, in a way favorable for binding. 3) This positive cooperative binding is achieved through steric conformational changes of the hemoglobin protein complex as discussed above, i.e. when one subunit protein in hemoglobin becomes oxygenated, this induces a conformational or structural change in the whole complex, causing the other subunits to gain an increased affinity for oxygen. As a consequence, the oxygen binding curve of hemoglobin is sigmoidal, or S-shaped, as opposed to the normal hyperbolic curve associated with noncooperative binding. Cooperative The ligand-binding sites are composed of both high- and low stability segments, so affinity for ligand is relatively low. (a) In the absence of ligand, the red segments are quite flexible and take up a variety of conformations, few of which facilitate ligand binding. The green segments are most stable in the low- affinity state. (b) The binding of ligand to one subunit stabilizes a high-affinity conformation of the nearby red segment (now shown in green), inducing a conformational change in the rest of the polypeptide. This is a form of induced fit. The conformational change is transmitted to the other subunit through protein-protein interactions, such that a higher-affinity conformation of the binding site is stabilized in the other subunit. A second ligand molecule can now bind to the second subunit, with a higher affinity than the binding of the first, giving rise to the observed positive cooperativity. Structural changes in a multisubunit protein undergoing cooperative binding to ligand. For example, in the upper left of the four hemes shown, oxygen binds causes the iron atom to move backward into the heme tuging the histidine residue closer pulls on the protein chain holding the histidine. A schematic visual model of oxygen binding process The binding and release of oxygen (shown now in green) illustrates the structural differences between oxy- and deoxyhemoglobin, respectively. The histidine which is pulled by motion of the iron atom, is shown here in yellow. Another view of how binding and release of ligands induces a conformational (structural) change in hemoglobin. Only one of the four heme groups is shown, Mechanism of the T-R transition in hemoglobin Some ion pairs that stabilize the T state of deoxyhemoglobin Several theories have been developed to describe allosteric transitions. They may be generally grouped into the following three classes: characterized by the co-existence of molecules with some subunits in the weak-binding state and some in the strong Sequential model, the prototype for the models that describe allosteric transitions Koshland, Nemethy, and Filmer (KNF model) The shift is a concerted (协同的) one Concerted model Monod, Wyman, and Changeux (MWC model) Adapted from G. K. Ackers et al., Science (1992) 255:54-63. the changes in tertiary structure that accompany oxygen binding can be tolerated up to a certain point before the T-R switch occurs. Specifically, whenever one site is occupied on each of the two - dimers, the molecule as a whole adopts the R quaternary structure Multistate model Hemoglobin binding O2 in lung (high O2) and lease it in tissue (low O2) A sigmoid (cooperative) binding curve. Cooperative binding renders hemoglobin more sensitive to the small differences in O2 concentration between the tissues and the lungs, allowing hemoglobin to bind oxygen in the lungs (where pO2 is high) and release it in the tissues (where pO2 is low). Allosteric Effecter: O2 A plot of log /(1-) versus log L is called a Hill plot The slope (斜率) of a Hill plot is denoted by nH, the Hill coefficient (希尔系数) Hill equation (希尔方程) 希尔方程和希尔系数 Theoretically nH=4 When nH1, there is no evident cooperativity. The maximum degree of cooperativity observed for hemoglobin corresponds approximately to nH3. Note that while this indicates a high level of cooperativity, nH is less than n, the number of O2-binding sites in hemoglobin. This is normal for a protein that exhibits allosteric binding behavior. Hill plots for the binding of oxygen to myoglobin and hemoglobin. Other Allosteric Effectors besides O2: 1, H+ 2, CO 3, CO2 4, BPG A pH drop in the blood in the capillaries lowers the oxygen affinity of hemoglobin, allowing even more efficient release of the last traces of oxygen. The response of hemoglobin to changes in pH is called the Bohr effect. The overall reaction may be written Hb-4O2 + nH+ Hb-nH+ + 4O2 (where n2) Physiologically, this reaction has two consequences: First, in the capillaries, hydrogen ions promote the release of O2 by driving the reaction to the right. Then, when the venous (静脉) blood recirculates to the lungs or gills (腮), the oxygenation has the effect of releasing the H+ by shifting the equilibrium to the left. This, in turn, tends to release CO2 from the bicarbonate dissolved in the blood by the reversal of the bicarbonate reaction: CO2 + H2O HCO3- + H+ The free CO2 can then be expired. the Bohr effect Hemoglobins oxygen-binding capacity is decreased in the presence of carbon monoxide because both gases compete for the same binding sites on hemoglobin, carbon monoxide binding preferentially in place of oxygen. The binding of oxygen is affected by molecules such as carbon monoxide (CO) (for example from tobacco smoking抽烟, car exhaust汽车尾气 and incomplete combustion in furnaces壁炉中的不充分燃烧). CO competes with oxygen at the heme binding site. Hemoglobin binding affinity for CO is 200 times greater than its affinity for oxygen, meaning that small amounts of CO dramatically reduce hemoglobins ability to transport oxygen. When hemoglobin combines with CO, it forms a very bright red compound called carboxyhemoglobin, which may cause the skin of CO poisoning victims to appear pink in death, instead of white or blue. When inspired air contains CO levels as low as 0.02%, headache and nausea occur; if the CO concentration is increased to 0.1%, unconsciousness will follow. In heavy smokers, up to 20% of the oxygen-active sites can be blocked by CO. Allosteric Effecter: CO, Competitive Hemoglobin also has competitive binding affinity for cyanide (CN-), sulfur monoxide (SO), nitrogen dioxide (NO2), and sulfide (S2-), including hydrogen sulfide (H2S). All of these bind to iron in heme without changing its oxidation state, but they nevertheless inhibit oxygen-binding, causing grave toxicity. CO 1) normal hemoglobin, 2) hemoglobin from an anemic (贫血的) individual with only 50% of her hemoglobin functional, and 3) hemoglobin from an individual with 50% of his hemoglobin subunits complexed with CO. Several oxygen- binding curves Carbon dioxide occupies a different binding site on the hemoglobin. Carbon dioxide is more readily dissolved in deoxygenated blood, facilitating its removal from the body after the oxygen has been released to tissues undergoing metabolism. This increased affinity for carbon dioxide by the venous (静脉) blood is known as the Haldane effect. Through the enzyme carbonic anhydrase, carbon dioxide reacts with water to give carbonic acid, which decomposes into bicarbonate and protons: CO2 + H2O H2CO3 HCO3- + H+ Allosteric Effecter: CO2 This reaction produces H+, contributing to the Bohr effect CO2 Oxygen Binding to Hemoglobin Is Regulated by 2,3- Bisphosphoglycerate (BPG) 2,3-二磷酸甘油酸 Allosteric Effecter: BPG Binding of 2, 3-bisphosphoglycerate to deoxyhemoglobin BPG binds at a site distant from the oxygen-binding site and regulates the O2-binding affinity of hemoglobin in relation to the pO2 in the lungs. Effect of BPG on the binding of oxygen to hemoglobin. 1) Hemoglobin binds to oxygen quite tightly when BPG is entirely absent, and the binding curve appears to be hyperbolic. 2) At sea level, hemoglobin is nearly saturated with O2 in the lungs, but only 60% saturated in the tissues, so the amount of oxygen released in the tissues is close to 40% of the maximum that can be carried in the blood. 3) At high altitudes, O2 delivery declines by about one-fourth, to 30% of maximum. An increase in BPG concentration, however, decreases the affinity of hemoglobin for O2, so nearly 40% of what can be carried is again delivered to the tissues. The BPG concentration in normal human blood is: about 5 mM at sea level and about 8 mM at high altitudes. Hemoglobin H (4) - A variant form of hemoglobin, formed by a tetramer of chains, which may be present in variants of thalassemia. Hemoglobin Barts (4) - A variant form of hemoglobin, formed by a tetramer of chains, which may be pres