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

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)

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

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

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

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

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

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.

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.

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.

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]

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

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.

1 atm = 101.325 kPa 13.3 kPa in lung 4 kPa in tissue 0.26 kPa

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

緊接的 遠端的

Evolution meaning……..

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

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

Hemoglobin binds oxygen cooperatively

Hemoglobin has two conformational states tense relaxed Oxygen binding state

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

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

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

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)

Hill equation slope

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

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

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

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)

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

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

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)

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

Sickle-cell Anemia It’s a genetic disease (each person has two alleles) Genetic variant in hemoglobin hemoglobin S Glu6 Val mutation in b chains (hydrophobic group) Forms immature red blood cells sickle cell Hemoglobin in blood is only half of the normal amount

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

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

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

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

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

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

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

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

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

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

b structure: Immunoglobulin fold

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

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

Antibodies bind tightly and specifically to antigen Kd ~ 10-10 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

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.

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

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)

Immunoblot assay

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

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

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

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

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

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

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

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

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

reference

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

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