1、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）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）。免疫记忆的分子基础。,配基(ligand): A molecule bound reversibly by a protein is called a ligand. A
2、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 t
3、o 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 af
4、fects 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 (
5、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 conformation
6、al 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
7、 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”,蛋白质的柔性 (Proteins are flexible),Grd19/SNX3,1 33 PX domain 158 162,phosphat
8、idylinositol-3-phosphatePtdIn(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.,2、Reversible Bi
9、nding 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
10、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 publish
11、ed 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 de
12、termined 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
13、. 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) Pe
14、rutz 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 res
15、ult 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 di
16、seases 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 humansHemoglobin variants are a part of the normal embryonic and fetal development, but
17、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
18、 (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% Hemoglobi
19、n 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 ser
20、ies 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 pr
21、oerythroblast (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
22、loses its RNA soon after entering the vasculature (脉管系统).,Synthesis,Role of the globins in oxygen transport and storage.,hemoglobin,myoglobin,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 proto
23、porphyrin (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 b
24、onds 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
25、于) to the plane.,Evolution of the globin genes,Evolutionary conservation of the globin 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
26、 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. Another view (derived fro
27、m 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
28、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
29、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 c
30、hange 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) T
31、he 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 conformati
32、onal 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 oxyge
33、n 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 ligan
34、d, 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 i
35、n 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 subuni
36、t. 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
37、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 dif
38、ferences 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,Mechani
39、sm 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 subuni
40、ts 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.
41、, 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,Multista
42、te 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 lung
43、s (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 plotThe 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 ev
44、ident 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 bind
45、ing 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 lowers the oxygen affinity of hemoglobin, allowing even more efficient release of the last traces of oxygen. The re
46、sponse of hemoglobin to changes in pH is called the Bohr effect. The overall reaction may be writtenHb-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,
47、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-
48、+ 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 b
49、y 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 am
50、ounts 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.,
