Lehninger Chapter 5: Protein-Ligand Interactions PDF

10/2/2023 Adapting the Hill Equation to the Binding of O2 to Hemoglobin (5-17) • Hill coefficient = nH = slope of a Hill plot – If nH = 1, ligand binding is not cooperative – nH > 1 indicates positive cooperativity – nH < 1 indicates negative cooperativity 52 52 Hill Plots for Myoglobin and Hem...

10/2/2023 Adapting the Hill Equation to the Binding of O2 to Hemoglobin (5-17) • Hill coefficient = nH = slope of a Hill plot – If nH = 1, ligand binding is not cooperative – nH > 1 indicates positive cooperativity – nH < 1 indicates negative cooperativity 52 52 Hill Plots for Myoglobin and Hemoglobin R * -> either know axis & Slope Interpretation binds Oz or it dusent -> the transition state shows I 53 53 Principle 5 (4 of 4) In a multisubunit protein, a conformational change in one subunit often affects the conformation of other subunits. 54 54 18 10/2/2023 Two Models Suggest Mechanisms for Cooperative Binding · both models make sense but neither are proven RRRR -ATT • MWC model = concerted model – all subunits in the same conformation "Functionally Identical" – ligand binds more tightly to the R state = -> - all same conformation & exist In the the same time subults transition • sequential model – each subunit can be in either conformation – equilibrium is altered as additional ligands are bound, progressively favoring the R state TTTT = TTTR Y TTRRI TRRRIRRRR 55 55 Concerted and Sequential Models Low affinity or inactive High affinity or active MWC model (concerted model) Sequential model 56 56 * Chot14 bioz) Red Nitebock( Hemoglobin Also Transports H+ and CO2 • hemoglobin carries two end products of cellular respiration: H+ and CO2 • carbonic anhydrase catalyzes the hydration of CO2 to bicarbonate: high (O2 & Law pH = Favor Tstate CO2 + H2O ⇌ H+ + HCO3– a stabolizes the T state lowers pH 57 57 19 10/2/2023 Principle 6 (3 of 3) Interactions between ligands and proteins may be regulated. 58 58 creat cen The Bohr Effect ↳ low • the structural effects of H+ and CO2 binding to affinity SAT hemoglobin favor the T state – the binding of H+ and CO2 is inversely related to the binding of O2 = * to , deliver Tstate and On to your tissethe .... PCOZ = * and pH = A ↳ Favors R State • Bohr effect = describes the effect of pH and [CO2] on the binding and release of O2 by hemoglobin ⑧+ HHB + O2 ⇌ HbO2 + H+ * HT, decrease PH Shift KAT , 59 59 The Effect of pH on O2 Binding to Hemoglobin saturations binding thate of • when [O2] is high, R State hemoglobin binds O2 and releases H+ -> • when [O2] is low, hemoglobin releases O2 and binds H+ ↳ ↑ PH PH * H binds to ACO2 4 Is the amino binding amino to one acids of the terminals 60 60 20 10/2/2023 Hemoglobin Binds CO2 • CO2 binding to hemoglobin is inversely related to binding of O2 – contributes to the Bohr effect by producing H+ -> when you bind you on Ht release 61 61 Oxygen Binding to Hemoglobin Is Regulated by 2,3-Bisphosphoglycerate (1 of 2) * - helps adapt IOW Oz in ↓ so • 2,3-bisphosphoglycerate (BPG): – example of heterotropic allosteric modulation – binds to a site distant from O2-binding site -middel of the pocket – greatly reduces the affinity of hemoglobin for oxygen to in to to high arbody the evelation alt increases decrease the high lungs BPG affitity so more to is 02 released tissue 62 62 Oxygen Binding to Hemoglobin Is Regulated by 2,3-Bisphosphoglycerate (2 of 2) • 2,3-bisphosphoglycerate (BPG): – example of heterotropic allosteric modulation – binds to a site distant from O2-binding site – greatly reduces the affinity of hemoglobin for oxygen HbBPG + O2 ⇌ HbO2 + BPG 63 63 21 10/2/2023 Effect of BPG on O2 Binding to Hemoglobin niberbolic • BPG increases at high altitudes are ↳sigmoid • hypoxia = lowered oxygenation of peripheral tissues – causes BPG increases 64 64 Binding of BPG to Deoxyhemoglobin • BPG binds to the cavity between the β subunits in the T state – cavity is lined with positively charged residues – BPG stabilizes the T state binds in pocket 65 65 Fetal Hemoglobin • fetus synthesizes α2γ2 hemoglobin -> – lower affinity for BPG than normal adult hemoglobin – higher affinity for O2 than normal adult hemoglobin - higher affinity fur low prevents Suite he CO2 66 66 22 10/2/2023 Sickle Cell Anemia Is a Molecular Disease of Hemoglobin • sickle cell anemia: – homozygous condition – single amino acid substitution (Glu6 to Val6) β chains produces a hydrophobic patch 67 67 Normal and Sickle Cell Hemoglobin • deoxygenated hemoglobin becomes insoluble and forms polymers that aggregate in tubular fibers • normal hemoglobin remains soluble upon deoxygenation 68 68 Sickle Cell Anemia • Physical exertion = weak, dizzy, short of breath – • • Heart murmurs and increased pulse Hemoglobin content in blood half the normal value (15-16 g /100 mL) Abnormally shaped cells block capillaries and interfere with normal organ function 69 69 23 10/2/2023 5.2 Complementary Interactions between Proteins and Ligands: The Immune System and Immunoglobulins 70 70 Principle 2 (3 of 4) A ligand binds a protein at a binding site that is complementary to the ligand in size, shape, charge, and hydrophobic or hydrophilic character. The interaction is specific: the protein can discriminate among the thousands of different molecules in its environment and selectively bind only one or a few types. A given protein may have separate binding sites for several different ligands. These specific molecular interactions are crucial in maintaining the high degree of order in a living system. 71 71 Immune Responses • immune response = coordinated set of interactions among many classes of proteins, molecules, and cell types – distinguishes molecular “self” from “nonself” and destroys “nonself” – eliminates viruses, bacteria, and other pathogens and molecules 72 72 24 10/2/2023 The Immune Response Includes a Specialized Array of Cells and Proteins • leukocytes = white blood cells, including macrophages and lymphocytes • The immune response consists of two complementary systems: – humoral immune system = directed at bacterial infections and extracellular viruses – cellular immune system = destroys infected host cells, parasites, and foreign tissues 73 73 The Humoral Immune Response • antibodies = immunoglobulins (Ig) = bind bacteria, viruses, or large molecules identified as foreign and target them for destruction – produced by B lymphocytes or B cells 74 74 The Cellular Immune Response • T lymphocytes = cytotoxic T cells (TC cells) – recognition of infected cells or parasites involves Tcell receptors on the surface of TC cells • helper T cells (TH cells) = produce soluble signaling proteins called cytokines – interact with macrophages – stimulate the selective proliferation of TC and B cells that can bind to a particular antigen (clonal selection) • memory cells = permit a rapid response to pathogens previously encountered 75 75 25 10/2/2023 Vaccines • often consists of weakened or killed virus or isolated proteins from a viral or bacterial protein coat • “teaches” the immune system what the viral particles look like, stimulating the production of memory cells 76 76 Principle 1 (3 of 4) The functions of many proteins involve the reversible binding of other molecules. A molecule bound reversibly by a protein is called a ligand. A ligand may be any kind of molecule, including another protein. The transient nature of protein-ligand interactions is critical to life, allowing an organism to respond rapidly and reversibly to changing environmental and metabolic circumstances. 77 77 Antigens and Haptens • antigen = molecule or pathogen capable of eliciting an immune response – can be a virus, a bacterial cell wall, or an individual protein or other macromolecule – antibodies or T-cell receptors bind to an antigenic determinant or epitope within the antigen • haptens = small molecules that can elicit an immune response when covalently attached to large proteins 78 78 26 10/2/2023 Antibodies Have Two Identical Antigen-Binding Sites • immunoglobulin G (IgG) = major class of antibodies – one of the most abundant blood serum proteins – 4 polypeptide chains: 2 heavy chains and 2 light chains – cleavage with protease papain releases the basal fragment Fc and two Fab branches (each with a single antigen-binding site) – constant domains contain the immunoglobulin fold structural motif 79 79 The Structure of Immunoglobulin G 80 80 The Variable Domain of Immunoglobulin G • heavy and light chains each have a variable domain – variable domains associate to create the antigen-binding site – allows formation of an antigenantibody complex 81 81 27 10/2/2023 Classes of Immunoglobulins • 5 classes in vertebrates: – characterized by heavy chain: • α for IgA • δ for IgD • ε for IgE • γ for IgG • μ for IgM 82 82 Structure of Immunoglobulins • IgD and IgE = similar in structure to IgG • IgM = monomeric, membrane-bound form or in a secreted form that is a cross-linked pentamer of this basic structure • IgA = monomer, dimer, or trimer IgM pentamer 83 83 Phagocytosis of Antibody-Bound Viruses by Macrophages • When Fc receptors bind an antibody pathogen complex, macrophages engulf the complex 84 84 28 10/2/2023 Principle 2 (4 of 4) A ligand binds a protein at a binding site that is complementary to the ligand in size, shape, charge, and hydrophobic or hydrophilic character. The interaction is specific: the protein can discriminate among the thousands of different molecules in its environment and selectively bind only one or a few types. A given protein may have separate binding sites for several different ligands. These specific molecular interactions are crucial in maintaining the high degree of order in a living system. 85 85 Antibodies Bind Tightly and Specifically to Antigen • induced fit = conformational changes in the antibody and/or antigen allow the complementary groups to interact fully • Kd values as low as 10–10 M 86 86 The Antibody-Antigen Interaction Is the Basis for a Variety of Important Analytical Procedures • two types of antibody preparations are used: – polyclonal antibodies • produced by injecting a protein into an animal • contain a mixture of antibodies that recognize different parts of the protein – monoclonal antibodies • Synthesized by a population of identical B cells (a clone) grown in cell culture • homogeneous, all recognizing the same epitope. 87 87 29 10/2/2023 Western Blots • immunoblot = Western blot assay = uses antibodies to detect a protein 88 ↑ student 88 presentation 5.3 Protein Interactions Modulated by Chemical Energy: Actin, Myosin, and Molecular Motors 89 89 The Major Proteins of Muscle Are Myosin and Actin -> remember a herativ temporar • arranged in filaments that undergo transient interactions and slide past each other to bring about contraction 90 90 30 10/2/2023 or-ATP snolignushaun wher gua Myosin Amino term • myosin (Mr 520,000) – 2 heavy chains and 4 light chains – forms a fibrous, left-handed coiled coil domain (tail) and a large globular domain (head) Hydraying site . Right handedLet madeil 91 carbox term . 91 Thick Filaments • thick filaments = rodlike structures of aggregated myosin - > core of contractive mit 92 92 Actin a globular • actin: in thin fillamints made from fractin – monomeric G-actin (Mr 42,000) associates to form a long polymer called F-actin ↳ Filliment , * right handed G-actin = O ... they form together to make F actin 93 93 31 10/2/2023 Thin Filaments • thin filaments = F-actin along with the proteins troponin and tropomyosin – assemble as successive monomeric actin molecules add to one end – upon addition each monomer binds and hydrolyzes ATP -> It takes tip actin to id (not for ) motion 94 94 mosine th actin binds to head of myosin = myofibrils : muscle Abe it Components of Muscle • each actin monomer in the thin filament binds to one myosin head group 95 95 Additional Proteins Organize the Thin and Thick Filaments into Ordered Structures • muscle fiber = large, single, elongated, multinuclear cell • each muscle fiber contains ~1,000 myofibrils, each consisting of thick and thin filaments and surrounded by sarcoplasmic reticulum -> receases control Cat to 96 96 32 10/2/2023 ~ simplest/smallest contractive cult The Structure of a Sarcomere • sarcomere = entire contractile unit – A band = stretches the length of the thick filament – I band = contains only thin filaments – Z disk = attachment site for thin filaments – M line = bisects the A band 97 zdisk intersects the centracted when - ↳ thin suand97 thick - 2 . Shrinks/HANow disn's also move together duser ↳ band I thich filaments we created by the association of many myosin * molecules * by Principle 1 (4 of 4) muscle centraction occurs thin sliding over thin . . . . The functions of many proteins involve the reversible binding of other molecules. A molecule bound reversibly by a protein is called a ligand. A ligand may be any kind of molecule, including another protein. The transient nature of protein-ligand interactions is critical to life, allowing an organism to respond rapidly and reversibly to changing environmental and metabolic circumstances. 98 98 Myosin Thick Filaments Slide along Actin Thin Filaments • myosin binds actin tightly when ATP is not bound to myosin ① -> ② -> • series of conformational changes due to binding, hydrolysis, and release of ATP and ADP causes muscle contraction infavorable ③ high energy state -> Pi= released . & glob reatactles down the Further change 99 99 33 10/2/2023 4 Steps -> 1. a. 1. unfavorable . ATP binds myosin; cleft in myosin molecule opens Disrupts actin-myosin interaction ATP is hydrolyzed; conformational change in protein to “high-energy” state a. b. Moves myosin head, changes orientation in relation to actin thin filament Myosin binds weakly to F-actin subunit closer to Z disk than what was just released 100 100 4 Steps Continued 3. Phosphate product of ATP hydrolysis is released a. Conformational change; myosin cleft closes (strengthening myosin-actin binding) 4. Conformation of myosin head returns to original resting state a. “Power stroke” b. Orientation pulls tail of myosin toward Z disk c. ADP released to complete the cycle 101 101 Tropomyosin regulates movement blocking myosin binding sites tropmyosin • tropomyosin = binds to the thin filament and blocks the myosin-binding sites by 102 102 34 10/2/2023 Troponin -> Bouncer • troponin = binds Ca2+ released from the sarcoplasmic reticulum, causes a conformational change, and exposes myosin-binding sites – subunit C binds Ca2+ – subunit I prevents binding of the myosin head to actin – subunit T links the troponin complex to tropomyosin cast then ti will troponin tropomyosin from tell reased be to stop blocking 103 103 Skeletal Muscle • • • Requires two types of molecular function: binding and catalysis Actin-myosin interaction is reversible and leaves participants unchanged Myosin is an actin-binding protein and an ATPase (enzyme) 104 104 35