中山大学生物化学课件

中山大学生物化学中山大学生物化学课件件4 Protein Function4.1 General features4.2 Oxygen-binding proteins4.3 Immunoglobulins 4.4 Muscle contractionliguofu4.1 General features1.Versatile in function2.Being hard to study 3.Function via interactionThe interaction with other molecule(ligand)is reversible.The interface between the binding site is complementary in structure,making such interaction highly specific.Structure dynamicness of a protein is usually essential for such interactions.liguofu4.1 General features4.2 Oxygen-binding proteins4.3 Immunoglobulins 4.4 Muscle contraction4 Protein FunctionliguofuQuantitative description of interaction(1)Protein+nLigand PLnliguofuQuantitative description of interaction(2)If kd is constantliguofuQuantitative description of interaction(3)If kd is not constantKdliguofuQuantitative description of interaction(4)liguofuQuantitative description of interaction(5)liguofuThe iron-porphyrin in hemoglobin accounts for the red color of blood,the copper-porphyrin in hemocyanin for blue color of blood,and the magnesium-porphyrin in chlorophyll is responsible for the green of plants.liguofuStructure of PorphyrinPyrrole ringMethene bridgeliguofuOxygen can be bound to a heme(1)Heme=Protoporphyrin IX+Fe2+None of the aa side chains in proteins is suited for reversible binding O2 Heme prosthetic groupTransition metals,Fe&Cu,have a strong tendency to bind O2needmorecommonlyliguofuOxygen can be bound to a heme(2)Heme=Protoporphyrin IX+Fe2+lFree Iron promotes the formation of highly reactive oxygen species such as hydroxyl radicals.lCoordinated N atoms help prevent the conversion of Fe2+to Fe3+liguofuMb has a single binding site for oxygenMyoglobin:153 residues,16700 DaHis93/F8His64/E7liguofuOxygen can be bound to a heme(3)lIn free heme molecules,reaction of oxygen at one of the two“open”coordination bonds of iron can result in irreversible conversion of Fe2+to Fe3+lIn heme-containing proteins,this reaction is prevented by sequestering the heme deep within a protein structure where acess to the two“open”coordination bonds is restricted.One of these two coordination bonds is occupied by a side-chain N of a His residue.NOCOAlsoliguofuQuantitative description of Mb binding O2liguofuProtein structure affects ligands bind(1)In free hemeorIn myoglobinIn free hemeIn myoglobin hemeorliguofu“Breathing”:Molecular motionsThe binding of O2 to heme in myoglobin depends on its“breathing”In myoglobin Protein structure affects ligands bind(2)liguofuHb is the most-studied and best-understood protein1.Hemoglobin was the first protein to be crystallized(in 1849);the first to be associated with a specific physiological function(around 1875);one of the first proteins to have its molecular weight determined correctly(64,500);the first eukaryotic messenger(mRNA)to be isolated and subsequently sequenced.the first eukaryotic protein to be synthesized in a cell-free system in vitro;the second protein having its 3-D structure determined(1969).liguofu2.The“red coloring matter”(hemoglobin)of animal blood could be brought to crystallization,with crystal forms characteristic of their biological origins(1840s-1860s).3.Reversible interconversion of oxyhemoglobin and deoxyhemoglobin was revealed by using spectroscope(1860s).4.Treatment of hemoglobin with acid gave a colorless albuminoid constituent(globin)and a red iron-containing material(by 1870).5.The structure of heme was elucidated to be a tetrapyrrol(porphyrin)derivative by chemical synthesis(Hans Fischer,1929).6.Similar tetrapyrrol derivatives are also used as prosthetic groups of other proteins(e.g.,the cytochromes that function in biological oxidation and photosynthesis!liguofuHb has four subunits Mr=64,500141 residues146 residuesliguofuHb subunits are structurally similar to myoglobin subunit lacks the short D helixD helixliguofuconserved in all known globinsconserved27 positions are identical,18%82143liguofu30 residuesInteractions in the four subunits19 residues19 residuesHydrophobic interactionsH bondsSalt bondsliguofuThis is called Tense state(deoxyHb)Some ion pairs are not shown hereIon pairs at the 1/2 and 2/1 interface(1)liguofuIon pairs at the 1/2 and 2/1 interface(2)Ion pairs stabilize the T state of deoxyHbliguofuQuantitative description of Hb binding O2（1）liguofuHill plotnH:hill coefficientQuantitative description of Hb binding O2（3）liguofuHb undergoes a structural change on binding O2(1)Tense state is more stable and thus the predominant conformation of deoxyHbRelaxed state is the predominant conformation of higher affinity to O2Binding O2Val E11liguofuIn the T state,the porphyrin is slightly puckered,causing the heme iron to protrude somewhat on the proximal His(His F8)side.The binding of O2 causes the heme to assume a more planar conformation,shifting the position of His F8 and F helix.Hb undergoes a structural change on binding O2(2)liguofuAllosteric protein is one in which the binding of a ligand to one site affects the binding properties of another site on the same protein.Modulator is a molecule that binds to an allosteric protein and affects its binding properties.Homotropic:the normal ligand and modulator are identical.Heterotropic:the normal ligand and modulator are not identical.Some proteins have two or more modulators and therefore can have both homotropic and heterotropic interactions.Hemoglobin binds oxygen cooperativelyliguofuTwo model for cooperative bindingConcerted ModelMWC ModelSequential ModelliguofuN-terminal residueCarbamino-terminal residueH+Hemoglobin transports CO2carbonic anhydrase15%to 20%80%to 85%liguofuHemoglobin also transports H+Bohr effectH+binds to:lNH of His146 in b bl4 N-terminal NH2lOthers:Arg,Lys liguofuO2 binding to Hb is regulated by BPG(1)liguofu O2 binding to Hb is regulated by BPG(2)liguofuBinding of BPG to deoxy-Hb stabilizes the T-state,thus lowers the O2 affinity of Hb.Only one BPG binds to each deoxy-Hb tetramerliguofuThe BPG binding pocket exists in the T-state,but disappears in the R-state of Hb.T-state(deoxy-Hb)R-state(oxy-Hb)Positively charged groups that BPG interacts withIn fetus hemoglobin(a2g g2),the conversion of b b-His143 to g g-ser143 results in the decrease of the affinity of BPGMutant Hbs provided a unique opportunity to study structure-function relationships in proteinsMany(500)Hb genetic variants,mostly(95%)with a single amino acid difference,have been identified in the human population.Most of such variation have a very minor effect on the protein structure and function.Some do cause debilitating diseases(e.g.,those from sickle cell anemia.)Deleterious mutations are mostly clustered about the heme pockets and in the vicinity of the -b-b contact region that is important in the allosteric transition.liguofuDistribution of mutations in human hemoglobin GluValSickle cell anemialiguofuliguofuSickle cell anemia is caused by a single amino acid replacement on the b b subunit:Glu6ValliguofuGluValliguofuliguofuElectron micrographof deoxy-Hb S fibersspilling out of a ruptured erythrocyte.The sickled cells are fragile.Their breakdown leads to an anemia that leaves the victim susceptible to infections and diseases.liguofuThe elongated cells tend to block capillaries,causing inflammation and considerable pain liguofuBy By Linus PaulingLinus Pauling in in 19491949.19031987NormalTraitPatient(Normal)(defective)liguofuThe HbS allele confers a small but significant resistance to malaria in heterozygous individualsThe HbS allele(i.e.,sickle cell trait)was found common in certain parts of Africa.Plasmodium(a protozoan causing malaria)increases the acidity of erythrocyte,favoring the formation of deoxy-HbS(the Bohr effect),thus probably allowing the spleen to preferentially remove the infected red cells.liguofu4.1 General features4.2 Oxygen-binding proteins4.3 Immunoglobulins4.4 Muscle contraction4 Protein FunctionliguofuliguofuStructure of immunoglobulin(1)liguofuStructure of immunoglobulin(2)H=Heavy chain L=Light chain C=Constant V=VariableCHO=carbohydrate liguofuStructure of immunoglobulin(3)liguofuStructure of Variable Region(1)Light ChainLight ChainHeavy ChainHeavy ChainCDRs:CDRs:complementarity-determining regionscomplementarity-determining regions Hypervariable loops Hypervariable loopsliguofuStructure of Variable Region(2)Heavy ChainHeavy ChainliguofuAntibody bind tightly&specifically to antigen(1)Small antigenSmall antigenliguofuSmall antigenSmall antigenAntibody bind tightly&specifically to antigen(2)Large antigen:lysozymeLarge antigen:lysozymeAntibody IAntibody IIVC1liguofuliguofuFive classes of immunoglobulins(1)liguofuliguofu抗抗体体的的作作用用：例例liguofuPolyclonal/Monoclonal antibodyliguofuliguofu4.1 General features4.2 Oxygen-binding proteins4.3 Immune proteins 4.4 Muscle contraction4 Protein FunctionliguofuMuscle structureliguofuMuscle structureliguofuMuscle contractionliguofuMuscle contractionliguofu2.Actin molecules3.Tropomyosin molecules4.Troponin moleculesCross bridges(head groups of myosin molecules)Actin Tropomyosin Troponin1.Myosin moleculehead groups Split by papainThin filament Thin filament Thick filament liguofu1.Myosin(1)(Two heavy chain)(four)Mr=540,000MrH=220,000MrL=20,000325 nmliguofu1.Myosin(2)liguofu1.Myosin(3)：S1 structureliguofu1.Myosin(3)：S1 structureliguofu2.G actin F actin(1)liguofu2.G actin F actin(2)=Ca2+or Mg2+liguofu2.G actin F actin(3)liguofuInteraction between myosin and F-actin liguofuStep 1Step 2The“power stroke”model of muscle contraction(ATP-consumingMotor)Two conformations of the cross-bridge were detected in insect muscle(1965)liguofuStep 3Step 4Step 1liguofuTropomyosin:lying along the groove in the F-actin helix.Troponin:TnC,TnI,TnTTnC:the Ca2+binding subunitTnI:the inhibitory subunitTnT:the Tm-binding subunitTm&Tn inhibit the binding of myosin heads to actin unless Ca2+is about 10-5M.In resting muscle,Ca2+is about 10-7M.The interaction between actin and myosin are regulated mainly by tropomyosin and troponin.liguofu3.Tropomyosin：coiled-coilliguofu4.Troponin(1)TnC:redTnI:light blueTnC binding region:blue TnT:yellowBound Ca2+:blackRH:regulatory headIT arm:TnI-TnT armliguofuCa24.Troponin(2)C-TnI:C-terminal region of TnIC-TnT:C-terminal region of TnTTnIreg:regulatory region of TnI:Regions where the structures are not well defined liguofuAnimation:process of muscle contractionliguofuCa2 approaches TroponinCa2 activates Tn to expose binding site for Myosin headMg2 approaches MhMh binds with Actin.Mg2 activates Mh causing release of Pi from ATP leaving ADP and causing the Mh to contractMg2and ADP released from Mh,ending its contractionMh releases from Actin.Ca2 released from Tn,allowing it to cover binding site.Next cycle.liguofuThe EndThe Endliguofu谢谢观赏！2020/11/587