1. Chapter 5 Protein Function
Dr. Rita P.-Y. Chen
陳佩燁博士
中央研究院生化所

2. Multifunctions of proteins
Catalyse biochemical reactions (enzyme)
Transduce signals (G-protein)
Control life and growth (hormone and receptor)
Provide protection (hair)
Provide mobility (muscle)
Transport materials (hemoglobin)
Recognize foreign material (immunoglobulin)
Induce disease (prion)

3. Protein-ligand binding
A molecule bound reversibly by a protein is called a ligand.
A ligand binds at a site on the protein called the binding site.
Ligand may be any kind of molecule.
The protein-ligand interaction is specific.
A protein may have separate binding sites for several ligands

4. Oxygen-binding protein
Oxygen is poorly soluble in aqueous solution. How can oxygen arrive in tissues?
Hemoglobin: transport oxygen and CO; tetramer, 2 a, 2 b subunits
Myoglobin: oxygen storage, single polypeptide of 153 amino acid residues

5. Why can they bind oxygen?
Prosthetic group: heme
Prosthetic group: A metal ion or an organic compound (other than an amino acid) that is covalently bound to a protein and is essentially to its activity

6. Heme: protoporphyrin, Fe2+
N atom has electron donating character which prevents the conversion of Fe2+ to Fe3+
Iron in Fe2+ state binds oxygen reversibly; in the Fe3+ state it does not bind oxygen

7. When oxygen binds, the electronic properties of heme iron change, so blood change color from dark purple to bright red

8. Hemoglobin subunits are structurally similar to Myoglobin
Hemoglobin has 2 a, 2 b subunits; less then half are sequence identical; but 3D-structures of a and b subunits are similar.
Only 27 amino acids are identical between myoglobin and hemoglobin subunits, but their structures are very similar.

10. Ka is an association constant
Ka has units of M-1;
The association constant provides a measure of the affinity of the ligand L for the protein.
a higher value of Ka corresponds to a higher affinity of the ligand for the protein.

11. The ratio of bound to free protein is directly proportional to the concentration of free ligand
When the concentration of the ligand is much greater than the concentration of ligand-binding sites, the binding of the ligand by the protein does not appreciably change the concentration of free (unbound) ligand —that is, [L] remains constant.

12. Now, use q(theta) to represent the ratio of ligand binding sites on the protein that are occupied by ligand
Because [PL] = Ka [L] [P]

13. Plot of q versus [L] Hyperbola 雙曲線
When half of sites are bound (q=0.5), 2[L] = [L] + 1/ Ka,
此時 [L] = 1/Ka = Kd Kd : dissociation constant 解離常數

14. Kd = 1/ Ka
Kd is given in units of molar concentration (M)
a lower value of Kd corresponds to a higher affinity of ligand for the protein.

15. 1 atm = kPa
13.3 kPa in lung
4 kPa in tissue
0.26 kPa

16. Why is CO toxic? To free heme Kd(CO) = 1/20000 Kd(O2)
To myoglobin Kd(CO) = 1/200Kd(O2) due to steric hinderance
How can gas enter the cavity?
Molecular motion of proteins
(breathing)
On a nanosecond time scale

17. 緊接的
遠端的

18. Evolution meaning……..

19. Hemoglobin: oxygen transportation
hemocytoblast
Cels with lots of hemoglobin
maturation
Cells lose intracellular organelles :
nucleus, mitochondria, endoplasmic reticulum
Erythrocytes (red blood cells)
Which survive only 120 days
Contain ~34 % hemoglobin

20. Hemoglobin 96 % saturated with O2
Each 100 ml blood releases 6.5 ml oxygen

21. Hemoglobin binds oxygen cooperatively

22. Hemoglobin has two conformational states
tense
relaxed
Oxygen binding state

24. T state is stabilized by ionic interactions, so T state is stable without oxygen binding

25. Why does oxygen binding induce T to R conformational change?
Puckered 縮攏
More planar
when heme binds O2 , porphyrin forms more planar conformation then shifts helix F ab subunits pairs slide past each other and rotate,narrowing the pocket between b subunits

26. High O2 affinity in lung, bind O2
Sigmoid binding curve
of hemoglobin
Low O2 affinity in tissue, release O2

27. When the normal ligand and modulator are identical homotropic;
Hemoglobin has 4 subunits, O2 binding to individual subunits can alter the affinity to O2 in adjacent subunits
Binding of a ligand to one site affects the binding properties of another site on the same protein allosteric protein
When the normal ligand and modulator are identical homotropic;
if not, heterotropic
Modulator (inhibitor or activator)

28. Hill equation
slope

29. Hill plot
Experimental determined slope reflects not the binding sites but the degree of interaction between them.
The slope of Hill plot is denoted by nH (Hill coefficient), representing degree of cooperativity
nH = 1 not cooperative
nH > 1 positive cooperative
nH < 1 negative cooperative

30. Two model explain hemoglobin cooperativity: MWC model (concerted model;all-or-none model), sequential model
一致的
連續的

31. Other modulators for hemoglobin : H+, CO2, 2,3-bisphosphoglycerate (BPG) reduce the affinity of hemoglobin for oxygen
Hemoglobin also carry 40 %H+ and 15-20% CO2 from tissue to lung and kidney to excrete them from body
CO2 is not very soluble
Catalyzed by carbonic anhydrase in earthrocyte
Bohr effect: the effect of pH and CO2 on binding and release of oxygen by hemoglobin

32. H+ binds to several residues , such as His146 (His HC3)
Low pH, low oxygen binding
T state is stabilized by ionic interactions. Protonated His helps stabilize T state (low oxygen affinity)

33. CO2 binds to the a-amino group at the N-terminal end of each subunit, forms carbaminohemoglobin
It can form additional salt bridges that help to stabilize T state and promote oxygen release

34. 2,3-bisphosphoglycerate (BPG)
BPG has high content in erythrocytes
BPG binds in the cavity between b subunits in the T state
R state
T state

35. BPG is important for delivering O2 to tissues when people is in high altitudes
If BPG is too high, it causes Hypoxia (組織缺氧)

36. Fetus needs to get oxygen from mother’s blood
Fetus has a2g2 hemoglobin (HbF)
a2g2 has lower affinity for BPG higher affinity for O2

37. Sickle-cell Anemia
It’s a genetic disease (each person has two alleles)
Genetic variant in hemoglobin hemoglobin S Glu Val mutation in b chains (hydrophobic group)
Forms immature red blood cells sickle cell
Hemoglobin in blood is only half of the normal amount

38. When hemoglobin S is deoxygenated, it becomes insoluble and forms polymers
The insoluble fibers are responsible to the sickle shape of erythrocyte
Sickle-cell anemia is more common in Africa result of natural selection of malaria

39. Protein-ligand interaction: Antibody and Antigen in immune system
Leukocytes (white blood cells)
macrophage
lymphocytes
T cells
B cells
Cytotoxic T cell (Tc cell)
Helper T cells
(TH cells)
Produce antibody (immunoglobulin; Ig)
Humoral immune system
Cellular immune system

40. Humoral (體液的)immune system is directed at bacterial infection and extracellular virus in body fluid, also respond to the proteins produces in these organism.
Cellular immune system destroys hosts infected by viruses, some parasites, and foreign tissues
與器官移植的排斥有關

41. Mature in bone marrow
Mature in thymus
HIV 會與 TH cells 結合,
破壞免疫能力

42. Any molecule or pathogen capable of eliciting an immune response is called an antigen
Part of the antigen which can be bound by antibody or T cell receptor is called antigenic determinant or epitope.
Molecules of Mr < 5000 are generally not antigenic. They can be covalently attached to large proteins to elicit an immune responses. They were called hapten .『免疫』附著素; 半抗原

43. MHC (major histocompatibility complex) proteins 敵我辨識
Made by Rita chen, IBC, Academia sinica
MHC (major histocompatibility complex) proteins 敵我辨識
Detection of protein antigens in the host is mediated by MHC
MHC proteins bind peptide fragments of protein digested in the cell and present them on the outside surface of the cell
Two classes of MHC proteins: class I, class II

44. Occur on the surface of vertebrate cells; polymorphic Therefore, it causes tissue rejection in organ transplanation
Occur on the surface of a few types of cells such as macrophage and B cells; polymorphic

45. The maturation of Tc cells in the thymus includes a stringent selection process that eliminates more than 95% of the developing Tc cells, including those that might recognize and bind class I MHC proteins displaying peptides from cellular proteins of the organism itself.
Each survived Tc cell has a single type of T-cell receptor that bind to one particular chemical structure
Human class I MHC protein binds antigen

46. Helper T cells (TH) TH cells works indirectly
TH cells stimulate the selectively proliferation of those Tc and B cells that bind to a particular antigen ----clonal selection
HIV binds TH cells AIDS

47. B cells produce 5 classes antibodies:IgA, IgD, IgE, IgG, IgM
2 heavy chains, 2 light chains
Papain cleavage gets Fab and Fc
Heavy chain has 5 types a,d,e,g,m for IgA, IgD, IgE, IgG, IgM, respectively
Light chain has types k and l
IgG

48. b structure:
Immunoglobulin fold

50. Phagocytosis of an antibody-bound virus by a macrophage
MHC I
Phagocytosis of an antibody-bound virus by a macrophage

51. IgA: found principally in secretions such as saliva, tears, milk; can be monomer, dimer, or trimer
IgM: first antibody produced by B cells; major antibody in the early stages of a primary immune response
IgG: major antibody in secondary immune response
IgE: allergic response; interact with basophil(呈鹼性染色顆粒的白血球細胞 ) and histamine-secreting cells (mast cell肥大細胞)
When antigen is bound by IgE, Fc of IgE binds to the Fc receptor of basophil or mast cell and induces them to secret histamine that causes dilation and increased permeability of blood vessels

52. Antibodies bind tightly and specifically to antigen
Kd ~ M
Binding specificity is determined by the residues in the variable domains of the heavy and light chains
Conformational change in antibody during binding antigen ----Induced fit

53. Polyclonal and monoclonal antibodies
Inject protein to an animal, obtained polycolonal antibodies are a mixture of antibodies that recognize different part of the protein. They are synthesized by many different B cells.
Monoclonal antibodies are synthesized by a population of identical B cells (a clone). They recognize the same epitope.

55. ELISA (enzyme-linked immunosorbent assay)
Rapid screening and quantification of the presence of an antigen in a sample

56. Test HSV antibody in blood
coat HSV antigen to well
Add blood sample (anti-HSV antibody)
Add anti-human IgG (linked with horseradish peroxidase)
Add substrate: yellow (positive signal)

57. Immunoblot assay

58. Protein interaction modulated by chemical energy: actin, myosin
Motor proteins:
Kinesin and dynein move along microtubules in cells, pulling along organelles or reorganizing chromosomes during division.
Helicase and polymerase move along DNA
Myosin and actin in muscle; actin and myosin make 80 % of protein mass of muscle

59. Myosin (Mr 540,000) has 6 subunits: 2 heavy chains (Mr 220,000) and 4 light chains (Mr 20,000)
Heavy chain: a-helix at C-terminal, forms left-handed coiled coil with another heavy chain
N-terminal of each heavy chain has ATPase activity and associates with 2 light chains

61. Myosin forms “thick filament” in muscle, one contractile unit has several hundred myosin

62. Right handed
Monomeric actin is called G-actin (globular actin, Mr 42,000)
G-actin will associate to form a long polymer called F-actin (filamentous actin)
“Thin filament” in muscle consists of F-actin, troponin and tropomyosin.
In the formation of F-actin, each G-actin binds ATP, then hydrolyzes it to ADP during association step. So in F-actin, each actin molecule is complexed to ADP

63. Each actin molecule in the thin filament can bind tightly and specifically to one myosin head group

65. Each muscle fiber is a single, very large, multinucleated cell
20 to 100 mm in diameter
It is formed from many cells fused together and often spanning the length of the muscle.
Each fiber has about 1000 myofibrils, consisting vast amount of regularly arrayed thick and thin filaments

66. When muscle contracts, the I bands narrow and the Z disks come closer
relaxed
contracted
M line is the high electron density region
Z disk is the anchor to which the thin filaments are attached

67. The thick and thin filaments are interleaved (交錯排列)
Each thick filament is surrounded by six thin filaments

68. There are other proteins involved in the sarcomere.
The actin filaments assembled with a-actinin, desmin, vimentin, nebulin(~7000 residues) attach to the Z disk
Except myosin, M line has paramyosin, C-protein, M-protein
Titin (26926 residues, the largest single polypeptide chain) links the thick filament to the Z disk. It regulates the length of sarcomere and prevents overtension of the muscle.
Nebulin and titin ----molecular ruler ----regulate the length of thin and thick filaments

69. reference

70. Myosin thick filaments slide along actin thin filaments and move toward Z disk
Each cycle generates 3 to 4 pN of force, moves 5 to 10 nm

71. How to regulate the contraction?
Tropomyosin forms a helix around the actin helix covering all the myosin binding sites.
Troponin is Ca2+ -binding protein. A nerve impulse causes Ca2+ release from sarcoplasmic reticulum. Ca2+ binds to troponin and causes conformational change in tropomyosin-troponin complex, exposing the myosin binding sites on the thin filaments