中山大学生物化学学习课件

1、liguofu 4 Protein Function 4.1 General features 4.2 Oxygen-binding proteins 4.3 Immunoglobulins 4.4 Muscle contraction liguofu 4.1 General features 1. Versatile in function 2. Being hard to study 3. Function via interaction The interaction with other molecule (ligand) is reversible. The interface be

2、tween the binding site is complementary in structure, making such interaction highly specific. Structure dynamicness of a protein is usually essential for such interactions. liguofu 4.1 General features 4.2 Oxygen-binding proteins 4.3 Immunoglobulins 4.4 Muscle contraction 4 Protein Function liguofu

3、 Quantitative description of interaction (1) Protein + nLigand PLn n n tiondisassocia PL LP k nn n n n PPL PL sitesbindingtotal occupiedsitesbinding n n nassociatio LP PL k d n n kL L liguofu Quantitative description of interaction (2) d n n kL L If kd is constant liguofu Quantitative description of

4、 interaction (3) d n n kL L If kd is not constant Kd liguofu d n n kL L d n k L 1 d kLnloglog) 1 log( Quantitative description of interaction (4) liguofu d kLnloglog) 1 log( Quantitative description of interaction (5) liguofu The iron-porphyrin in hemoglobin accounts for the red color of blood, the

5、copper-porphyrin in hemocyanin for blue color of blood, and the magnesium-porphyrin in chlorophyll is responsible for the green of plants. liguofu Structure of Porphyrin Pyrrole ring Methene bridge liguofu Oxygen can be bound to a heme (1) Heme = Protoporphyrin IX + Fe2+ None of the aa side chains i

6、n proteins is suited for reversible binding O2 Heme prosthetic group Transition metals, Fe 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 b

7、e 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). liguofu 2. The “red coloring matter” (hemoglobin) of animal blood could be brought to crystallization, with crystal for

8、ms 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 187

9、0). 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!

10、liguofu Hb has four subunits Mr = 64,500 141 residues 146 residues liguofu Hb subunits are structurally similar to myoglobin subunit lacks the short D helix D helix liguofu conserved in all known globins conserved 27 positions are identical, 18% 82 143 liguofu 30 residues Interactions in the four su

11、bunits 19 residues 19 residues Hydrophobic interactions H bonds Salt bonds liguofu This is called Tense state (deoxyHb) Some ion pairs are not shown here Ion pairs at the 1/2 and 2/1 interface (1) liguofu Ion pairs at the 1/2 and 2/1 interface (2) Ion pairs stabilize the T state of deoxyHb liguofu Q

12、uantitative description of Hb binding O2（1） liguofu Hill plot nH: hill coefficient Quantitative description of Hb binding O2（3） liguofu Hb undergoes a structural change on binding O2 (1) Tense state is more stable and thus the predominant conformation of deoxyHb Relaxed state is the predominant conf

13、ormation of higher affinity to O2 Binding O2 Val E11 liguofu In 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

14、 helix. Hb undergoes a structural change on binding O2 (2) liguofu Allosteric 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

15、. 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 cooperatively liguofu Two model for co

16、operative binding Concerted Model MWC Model Sequential Model liguofu N-terminal residue Carbamino- terminal residue H+ Hemoglobin transports CO2 carbonic anhydrase 15% to 20% 80% to 85% liguofu Hemoglobin also transports H+ Bohr effect H+ binds to: lNH of His146 in l4 N-terminal NH2 lOthers: Arg, Ly

17、s liguofu O2 binding to Hb is regulated by BPG (1) liguofu O2 binding to Hb is regulated by BPG (2) liguofu Binding of BPG to deoxy-Hb stabilizes the T- state, thus lowers the O2 affinity of Hb. Only one BPG binds to each deoxy-Hb tetramer The BPG binding pocket exists in the T-state, but disappears

18、 in the R-state of Hb. T-state (deoxy-Hb) R-state (oxy-Hb) Positively charged groups that BPG interacts with In fetus hemoglobin (a2g2), the conversion of His143 to gser143 results in the decrease of the affinity of BPG liguofu Mutant Hbs provided a unique opportunity to study structure-function rel

19、ationships in proteins Many (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 c

20、ell anemia.) Deleterious mutations are mostly clustered about the heme pockets and in the vicinity of the contact region that is important in the allosteric transition. liguofu Distribution of mutations in human hemoglobin GluVal Sickle cell anemia liguofu liguofu Sickle cell anemia is caused by a s

21、ingle amino acid replacement on the subunit: Glu6Val liguofu GluVal liguofu liguofu Electron micrograph of deoxy-Hb S fibers spilling 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. liguof

22、u The elongated cells tend to block capillaries, causing inflammation and considerable pain liguofu By Linus Pauling in 1949. 1903 1987 Normal TraitPatient (Normal) (defective) liguofu The HbS allele confers a small but significant resistance to malaria in heterozygous individuals The HbS allele (i.

23、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. liguofu 4.1 Gen

24、eral features 4.2 Oxygen-binding proteins 4.3 Immunoglobulins 4.4 Muscle contraction 4 Protein Function liguofu liguofu Structure of immunoglobulin (1) liguofu Structure of immunoglobulin (2) H = Heavy chain L = Light chain C = Constant V = Variable CHO = carbohydrate liguofu Structure of immunoglob

25、ulin (3) liguofu Structure of Variable Region (1) Light Chain Heavy Chain CDRs: complementarity-determining regions Hypervariable loops liguofu Structure of Variable Region (2) Heavy Chain liguofu Antibody bind tightly & specifically to antigen(1) Small antigen liguofu Small antigen Antibody bind ti

26、ghtly & specifically to antigen(2) Large antigen: lysozyme Antibody IAntibody II V C1 liguofu liguofu Five classes of immunoglobulins (1) liguofu liguofu 抗体的作用：例 liguofu Polyclonal /Monoclonal antibody liguofu liguofu 4.1 General features 4.2 Oxygen-binding proteins 4.3 Immune proteins 4.4 Muscle co

27、ntraction 4 Protein Function liguofu Muscle structure liguofu Muscle structure liguofu Muscle contraction liguofu Muscle contraction liguofu 2.Actin molecules 3.Tropomyosin molecules 4.Troponin molecules Cross bridges (head groups of myosin molecules) Actin Tropomyosin Troponin 1. Myosin molecule he

28、ad groups Split by papain Thin filament Thin filament Thick filament liguofu 1.Myosin (1) (Two heavy chain) (four) Mr = 540, 000 MrH = 220, 000 MrL = 20, 000 325 nm liguofu 1.Myosin (2) liguofu 1.Myosin (3)：S1 structure liguofu 1.Myosin (3)：S1 structure liguofu 2.G actin F actin (1) liguofu 2.G acti

29、n F actin (2) = Ca2+ or Mg2+ liguofu 2.G actin F actin (3) liguofu Interaction between myosin and F-actin liguofu Step 1 Step 2 The “power stroke” model of muscle contraction (ATP-consuming Motor) Two conformations of the cross-bridge were detected in insect muscle (1965) liguofu Step 3 Step 4 Step

30、1 liguofu Tropomyosin: lying along the groove in the F-actin helix. Troponin: TnC, TnI, TnT TnC: the Ca2+binding subunit TnI: the inhibitory subunit TnT: the Tm-binding subunit Tm & Tn inhibit the binding of myosin heads to actin unless Ca2+ is about 10-5M. In resting muscle, Ca2+ is about 10-7M. Th

31、e interaction between actin and myosin are regulated mainly by tropomyosin and troponin. liguofu 3. Tropomyosin：coiled-coil liguofu 4. Troponin (1) TnC: red TnI: light blue TnC binding region: blue TnT: yellow Bound Ca2+: black RH: regulatory head IT arm: TnI-TnT arm liguofu Ca2 4. Troponin (2) C-Tn

32、I: C-terminal region of TnI C-TnT: C-terminal region of TnT TnIreg: regulatory region of TnI :Regions where the structures are not well defined liguofu Animation: process of muscle contraction liguofu Ca2 approaches Troponin Ca2 activates Tn to expose binding site for Myosin head Mg2 approaches Mh M

33、h binds with Actin. Mg2 activates Mh causing release of Pi from ATP leaving ADP and causing the Mh to contract Mg2and ADP released from Mh, ending its contraction Mh releases from Actin. Ca2 released from Tn, allowing it to cover binding site. Next cycle. liguofu The End liguofu 2. The “red coloring

34、 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 al