血紅蛋白和免疫球蛋白

1、1、Concepts 基本概念2、Reversible Binding of a Ligand to a Protein: 肌紅蛋白和血紅蛋白3、Complementary Interactions between Proteins and Ligands: 免疫系統(tǒng)和免疫球蛋白,§2.4 蛋白質(zhì)的結(jié)構(gòu)和功能,本次作業(yè)（第三次作業(yè)）海拔高度調(diào)控別構(gòu)效應(yīng)子BPG濃度的分子基礎(chǔ)（或可以理解為海拔高度如何決定

2、代謝產(chǎn)物BPG的濃度）。免疫記憶的分子基礎(chǔ)。,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 bindi

3、ng 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 prote

4、in 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 (誘導(dǎo)契合).,In a multisubunit protein, a con

5、formational change in one subunit often affects the conformation of other subunits.,Intermolecular signal transduction,結(jié)合常數(shù),解離常數(shù),低解離常數(shù)與親和層析,Enzymes represent a special case of protein function. Enzymes bind and chemicall

6、y 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 (催化位點(diǎn)) or

7、active site (活性位點(diǎn)).,底物和活性位點(diǎn),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

8、the protein that affect the binding of the first ligand. (for example, the case of BPG)Allosteric (變構(gòu)效應(yīng)) - an effect that affects the activity of one part of an enzyme (such as an active site) by the binding of a molec

9、ule at a different site (regulatory site) at a different location on the enzyme.,變構(gòu)效應(yīng)/別構(gòu)效應(yīng),Changes in conformation may be subtle, reflecting molecular vibrations and small movements of amino acid residues throughout the

10、protein. A protein flexing (撓動(dòng)) in this way is sometimes said to “breathe”,蛋白質(zhì)的柔性 (Proteins are flexible),Grd19/SNX3,,,1 33 PX domain 158 162,,phosphatidylinositol-3-pho

11、sphatePtdIn(3)P磷脂酰肌醇-3-磷酸,Kd=0.15~0.5 µM,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

12、changes are frequently essential to a protein’s function.,2、Reversible Binding of a Ligand to a Protein: 肌紅蛋白和血紅蛋白,血紅蛋白: hemoglobin-oxygen transport protein (α2β2 in complex with 4 hemes)肌紅蛋白: myoglobin-oxygen st

13、orage 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 ligan

14、d 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 (血紅素),In 1840, the oxygen-carrying protein haemoglobin was discovered by Hünefe

15、ld.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 evap

16、oration 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 determine

17、d 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.

18、 and Phillips DC. (1958). "A Three-Dimensional Model of the Myoglobin Molecule Obtained by X-Ray Analysis". Nature 181 (4610): 662–666. 2) Perutz, M.F.; Rossmann, M.G.; Cullis, A.F.; Muirhead, H.; Will, G.; No

19、rth, A.C.T. (1960). "Structure of H". Nature 185 (4711): 416–422. 3) Perutz MF (November 1960). "Structure of hemoglobin". Brookhaven symposia in biology 13: 165–83.,Research history,1) The sequences

20、 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 m

21、utant 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 l

22、evel. 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 pr

23、oduce anemia.,Genetics,Types in humansHemoglobin 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

24、. 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 (ζ2ε2) Gower 2 (α2ε2) (PDB

25、1A9W) Hemoglobin Portland (ζ2γ2) In the fetus:Hemoglobin F (α2γ2) (PDB 1FDH) In adults:Hemoglobin A (α2β2) (PDB 1BZ0) -The most common with a normal amount over 95% Hemoglobin A2 (α2δ2) -δ chain synthesis begins

26、late in the third trimester and in adults, it has a normal range of 1.5-3.5% Hemoglobin F (α2γ2) - In adults Hemoglobin F is restricted to a limited population of red cells called F-cells. However, the level of Hb F can

27、 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

28、 series of steps in the mitochondria (線粒體) 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 it

29、s early development from the proerythroblast (原成紅細(xì)胞) to the reticulocyte (網(wǎng)織紅細(xì)胞) in the bone marrow (骨髓). 4) The nucleus is lost in mammalian (哺乳動(dòng)物) red blood cells, but not in birds and many other species. Even after t

30、he 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 (脈管系統(tǒng)).,Synthesis,Role of the globins in oxygen transport

31、and storage.,hemoglobin,myoglobin,,,The iron atom of heme (亞鐵血紅素) 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

32、 one example, consist of four pyrrole (吡咯) rings linked by methene (亞甲基) bridges, with substitutions at one or more of the positions denoted X.,Heme (亞鐵血紅素),This view shows the two coordination bonds to Fe2＋ perpendicula

33、r 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

34、 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,Evolutionary conservation of the globi

35、n folding pattern,The structure of myoglobin,Myoglobin,Oxygen binds to heme with the O2 axis at an angle, a binding conformation readily accommodated by myoglobin. Carbon monoxide binds to free heme with the CO axis per

36、pendicular(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. T

37、his effect weakens the binding of CO to myoglobin. 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

38、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 op

39、ening 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

40、to let it escape,撥浪鼓,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 conformat

41、ion is called relaxed (松弛的) (R). The tighter conformation is called tense (緊張的) (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 depart

42、ed, 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

43、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

44、 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 o

45、r 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

46、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,

47、 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

48、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 transmi

49、tted 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

50、, 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

51、 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 oxy

52、gen 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,

53、 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 hemog

54、lobin,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 b

55、y 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 m

56、odel),The shift is a concerted (協(xié)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

57、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 α-β dimers, the molecule as a whole adopts the R quaternary structure,Multistate model,

58、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 betwe

59、en 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 [θ/(1-θ)] versus log [L] is called

60、 a Hill plotThe slope (斜率) of a Hill plot is denoted by nH, the Hill coefficient (希爾系數(shù)),Hill equation (希爾方程),希爾方程和希爾系數(shù),Theoretically nH=4,When nH＝1, there is no evident cooperativity. The maximum degree of cooperativ

61、ity observed for hemoglobin corresponds approximately to nH＝3. 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 t

62、hat exhibits allosteric binding behavior.,Hill plots for the binding of oxygen to myoglobin and hemoglobin.,Other Allosteric Effectors besides O2:1, H+ 2, CO3, CO2 4, BPG,A pH drop in the blood in the capillaries lo

63、wers 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 writtenHb-4

64、O2 + nH+ Hb-nH+ + 4O2 (where n>2) 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 veno

65、us (靜脈) 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

66、blood by the reversal of the bicarbonate reaction: CO2 + H2O HCO3- + H+The free CO2 can then be expired.,the Bohr effect,Hemoglobin's oxygen-binding capacity is decreased in the presence of carbon monoxide becau

67、se 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 tob

68、acco smoking抽煙, car exhaust汽車尾氣 and incomplete combustion in furnaces壁爐中的不充分燃燒). CO competes with oxygen at the heme binding site. Hemoglobin binding affinity for CO is 200 times greater than its affinity for oxygen, m

69、eaning that small amounts of CO dramatically reduce hemoglobin's ability to transport oxygen. When hemoglobin combines with CO, it forms a very bright red compound called carboxyhemoglobin, which may cause the skin o

70、f 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 wil