III Structure, Catalysis and Information Pathways 3. Amino Acids, Peptides, and Proteins 4. The Three-Dimensional Structure of Proteins 5. Protein Functions 6. Enzymes 7. Carbohydrates and Glycobiology 8. Lipids 9. Biological Membranes and Transport 10. Biosignaling 11. Nucleotides and Nucleic Acids 12. Genes and Chromosomes 13. DNA Metabolism 14. RNA Metabolism 15. Protein Metabolism

Ligand; A molecule bound reversibly by a protein is called a ligand. Binding site; A ligand binds at a site on the protein, which is complementary to the ligand in size, shape, charge and hydrophobic or hydrophilic character. Induced fit; The binding of a protein and ligand is often coupled to a conformational change in the protein that makes the binding site more complementary to the ligand, permitting tighter binding. This structural adaptation is called induced fit. Substrate; The molecule acted upon by enzyme is called to be reaction substrate.

What are the properties of protein function? 1. Reversible Binding of a Protein to a Ligand Oxygen-Binding Proteins 2. Complementary Interactions between Proteins and Ligands Immune System and Immnunoglobulins 3. Protein Interactions Modulated by Chemical Energy Actin, Myosin, and Molecular Motors

Myoglobin and Hemoglobin

Myoglobin Has a Single Binding Site for Oxygen Mb; Mr, 16,700; 153 a.a., one heme; 8 α-helical segments (A –H), Oxygen storage diffusion, Found primarily in muscle. Lower His bonds covalently to iron(II) Oxygen coordinates to sixth site on iron and the upper His acts as a “gate” for the oxygen.

Protein-Ligand Interactions Can Be Described Quantitatively P + L PL [PL] [P][L] Association constant Ka; Dissociation constant Kd; Ka = [PL] [P][L] Kd = Binding sites occupied Total binding sites = [L] + 1 [L] + Kd [L] Ka θ [PL] + [P] [PL]

θ = [L]/([L] + Kd) When [L] is equal to Kd, half of the ligand-binding sites are occupied. As [L] falls below Kd, progressively less of the protein has ligand bound to it. In order for 90% of the availble ligand-binding sites to be occupied, [L] must be nine times grater than Kd.

The more tightly a protein binds a ligand, the lower the concentration of ligand required for half the binding sites to be occupied, and thus the lower the value of Kd.

Protein Structure Affects How Ligands Bind The binding of a ligand to a protein in organisms is rarely as simple as above equations, The interaction is greatly affected by protein structure and is often accompanied by conformational changes, called molecular breathing. If the protein is rigid, the ligand could not enter or leave the binding site at a measurable rate. However, the molecular breathing of protein makes its way in and out more easier.

CO (carbon monoxide) binds to free heme molecules over 20,000 times better than does O2 , but binds only about 200 times better when the heme is bound in myoglobin. Distal His

Oxygen Is Transported in Blood by Hemoglobin Nearly all the oxygen carried by whole blood in animals is bound and transported by hemoglobin in erythrocytes (red blood cells).

Erythrocytes are formed from precursor stem cells (hemocytoblasts) Erythrocytes are formed from precursor stem cells (hemocytoblasts). Their main function is to carry hemoglobin which is dissolved in cytosol at a very high concentration (-34%). In the maturation process, the stem cell produces daughter cells that form large amounts of hemoglobin and then lose their intracelluar organelles. Thus, erythrocytes are vestigial cells, unable to reproduce and with short life-time (120 days in humans).

Hemoglobin Subunits Are Structurally Similar to Myoglobin Hb; Mr, 64,500;Tetrameric protein; four heme groups, two α–chains (141 a.a.for each) and two β–chains (146 a.a. for each). Structures are similar to that of myoglobin. Found in erythrocytes; Transport oxygen

Hemoglobin Subunits Are Structurally Similar to Myoglobin

Hemoglobin Subunits Are Structurally Similar to Myoglobin Hydrophobic interaction between unlike subunits; α1β1 interface (and α2β2) involves over 30 residues. α1β2 (and α2β1) interface involves 19 residues.

Hemoglobin Undergoes a Structural Change on Binding Oxygen Ionic interaction between the subunits

Hemoglobin Undergoes a Structural Change on Binding Oxygen R state (relaxed); oxygen binding T state (tense); oxygen absence

Hemoglobin Undergoes a Structural Change on Binding Oxygen

Hemoglobin Undergoes a Structural Change on Binding Oxygen

Hemoglobin Binds Oxygen Cooperatively Allosteric protein; is one in which the binding of a ligand to one site effects the binding properties of another site on the same protein. Sigmoid binding curve Allosteric proteins are those having conformations (interconvert between more-active and less-active) induced by the binding of ligands referred to as modulators.

Cooperative Ligand Binding Can Be Described Quantitatively Archibald Hill, 1910 Hill plot; Log[θ/(1-θ) = nlog[L] – logKd logKd = nlog[L50] Hill coefficient; nH nH equal 1, ligand binding is not cooperative. nH greater than 1, ligand binding is positive cooperative. nH less than 1, ligand binding is negative cooperative.

Two Models Suggest Mechanisms for Cooperative Binding Concerted Model Sequential Model

Chloride and Bicarbonate Are Cotransported across the Erythrocyte Membrane Waste CO2 released from respiring tissues into the blood plasma enters the erythrocyte, where it is converted into bicarbonate (HCO3 ) by the enzyme carbonic anhydrase (脱水酶). The HCO3 reenters the blood plasma for transport to the lungs. In the lungs, HCO3 reenters the erythrocyte and is converted to CO2, which is eventually exhaled. For this shuttle to be effective, very rapid movement of HCO3 across the erythrocyte membrane is required.

Hemoglobin Also Transports H+ and CO2 Hb bind CO2; CO2 binds as a carbmate group to the amino group at the amino-terminal end of each subunit chain, forming carbaminoHb. The reaction produces H+, and help to stabilize the T state and promote the release of oxygen.

Hemoglobin Also Transports H+ and CO2 Bohr effect; At the relatively low pH and high CO2 concentration of peripheral tissues, the affinity of Hb for oxygen decreases as H+ and CO2 bound, and O2 is released to the tissues. Conversely, in the lung, as CO2 is excreted and the blood pH rises, the affinity of Hb for oxygen increases and Hb binds more O2 for transporting.

Hemoglobin Also Transports H+ and CO2 Hb bind proton; H+ binds to any of several amino acid residues in the protein. A major contribution to the Bohr effect is made by His146 of β subunits, When protonated, forms a ion pairs with Asp94, that help stabilize deoxyHb in the T state. HHb+ + O2 Hb+ + H+

Animation: Concerted allostery Sequential allostery

Oxygen Binding to Hemoglobin Is Regulated by 2,3-Bisphosphoglycerate The interaction of 2,3-bisphosphoglycerate (BPG) with Hb modulates O2 binding to Hb to adapt environmental change on pO2 or O2 concentration. BPG binds at a distant from the O2-binding site and regulates the O2-binding affinity of Hb in relation to the pO2 in the lungs. 2,3-bisphosphoglycerate (BPG); Is a reducer of affinity of Hb for oxygen HbBPG + O2 HbO2 + BPG

Oxygen Binding to Hemoglobin Is Regulated by 2,3-Bisphosphoglycerate BPG binds to Hb in the cavity between the β subunits in the T state. This cavity is lined with positively charged amino acid residues that interact with the negatively charged groups of BPG. BPG lowers Hb’s affinity for oxygen by stabilizing the T state. The transition to the R state narrows the binding pocket for BPG, precluding BPG binding.

Effect of BPG on the binding of oxygen to hemoglobin

Sickle-Cell Anemia Is a Molecular Disease of Hemoglobin Sickle-cell anemia is a genetic disease in which an individual has inherited the allele for sickle-cell Hb from both parents. The erythrocytes of these individuals are fewer and also abnormal. In addition to an unusually large number of immature cell, the blood contains many long, thin, crescent-shaped erythrocytes that look like the blade of a sickle.

Sickle-Cell Anemia Is a Molecular Disease of Hemoglobin Glu6--Val6 in the two β chains

Protein Function *** Reversible Binding of a Protein to a Ligand: Oxygen-Binding Proteins *** Complementary Interactions between Proteins and Ligands: The Immune System and Immnunoglobulins ***Protein Interactions Modulated by Chemical Energy: Actin, Myosin, and Molecular Motors

Immune system; vertebrates distinguish molecular self from nonself and then destroying those entities identified as nonself (humoral and cellular immune system). Each recognition protein of the immune system specifically binds some particular chemical structure, distinguishing it from virtually all others.

Antibodies Play the Central Functions in the Humoral Immune System Antibodies (immunoglobulins, Ig); The glycoproteins at the heart of the humoral immune response are soluble and bind bacteria, viruses, or large molecules identified as foreign and target them for destruction. Ig are produced by B lymphocytes and making up 20% of blood protein. Antigen; Any molecule or pathogen capable of eliciting an immune response is called antigen. An antigen may be a virus, a bacteria cell wall, or an individual protein or other macromolecule (Mr > 5,000).

Immunoglobulin (Ig) any member of a group of proteins occurring in higher animals as major components of the immune system. They are produced by cells of the lymphocyte series, and virtually all possess specific antibody activity. Each Ig molecule essentially comprise four polypeptide chains, two identical heavy chains and two identical light chains, linked together by disulfide bonds. There are five classes, IgA, IgD, IgE, IgG, and IgM.

Antibodies Have Two Identical Antigen-Binding Sites Immunoglobulin G (IgG) is the marjor class of antibody molecule and one of the most abundant proteins in the blood serum. IgG has four polypeptide chains: two large ones, called heavy chains, and two light chains, linked by noncovalent and disulfide bonds into a complex of Mr 150,000. Fc; crystallize fragment Fab; antigen-binding Fragment

Antibodies Have Two Identical Antigen-Binding Sites

Antibodies Have Two Identical Antigen-Binding Sites Induced fit Binding of IgG to an antigen

IgM pentamer Antibodies Have Two Identical Antigen-Binding Sites Five Immunoglobulin Classes; IgA, IgD, IgE, IgG, and IgM have a characteristic type of heavy chain, denoted and α,δ,ε,γ, and μ, respectively. Two types of light chain κand λ occur in all classes . IgD and IgE are similar to that of IgG, IgA was found in secretions such as saliva, tears and milk, can be a monomer, dimer or trimer. IgM occurs in B cell membrane-bound form with monomer and/or secreted form with pentamer. IgM pentamer

Antibodies Have Two Identical Antigen-Binding Sites Phagocytosis of an antibody-bound virus by a macrophage

Antibodies Bind Tightly and Specifically to Antigen Induced fit in the binding of an antigen (HIV) to IgG

The Antibody-Antigen Interaction Is the Basis for a Variety of Important Analytical Procedures Polyclonal antibodies; are those produced by many different B clles responding to one antigen, such as a protein injected into an animal. Cell in the population of B cells produce antibodies that bind specific, different epitopes within the antigen. Thus, polyclonal preparations contain a mixture of antibodies that recognize different parts of the protein. Monoclonal antibodies; are synthesized by a population of identical B cells (a clone) grown in cell culture. These antibodies are homogeneous, all recognizing the same epitope.

Detection of Pregnancy by Antibody hCG; human chorionic gonadotropin

The Antibody-Antigen Interaction Is Used to Localize Proteins

The Antibody-Antigen Interaction Is Used to Localize Proteins

ELISA (enzyme-linked immunosorbent assay) allows for rapid screening and quatification of certin protein in a sample

Purification of Protein by Antibody-Affinity Chromatography

Immunoblot assay (Western Blotting) Western Blotting allows the detection of a minor component in a sample and provide an approximation of its molecular weight. Immunoblot assay (Western Blotting) 1. Separation of proteins by gel electrophoresis 2. Transfer the separated proteins to a membrane 3. Membrane blocking with nonspecific protein 4. Incubate with primary antibody 5. Incubate with secondary antibody (antibody-enzyme complex) 6. Add substrate 7. Formation of colored

Co-immunoprecipitation Working with Protein-Protein Reactions Co-immunopreciptation Co-immunoprecipitation binding wash elution Y

Protein Function *** Reversible Binding of a Protein to a Ligand: Oxygen-Binding Proteins *** Complementary Interactions between Proteins and Ligands: The Immune System and Immnunoglobulins *** Protein Interactions Modulated by Chemical Energy: Actin, Myosin, and Molecular Motors

The Major Proteins of Muscle Are Myosin and Actin Myosin (Mr. 540 kD): has six subunits with two heavy chains and four light chains. At its amino terminus, each heavy chain has a large globular domain containing a ATP hydrolytic sit. The light chains associated with the globular domains. In muscle cells, molecules of myosin aggregate to form a rodlike structures (Thick filaments). These structures serve as the core of the constractile unit.

The Major Proteins of Muscle Are Myosin and Actin Actin the monomeric actin called G-actin (Mr. 42 kD, associate to form a long polymer called F-actin. In muscle cells, thin filament consists of F-actin (long polymer), along with the proteins troponin and tropomyosin.

Additional Proteins Organize the Thin and Thick Filaments into Ordered Structures

Additional Proteins Organize the Thin and Thick Filaments into Ordered Structures

ATP binds to myosin ATP is then hydrolyzed. The phosphate product of ATP is released from myosin. 4. ADP is then released to complete the cycle.

Protein Functions (in a biological process) 1. Catalysis: 2. Structure: 3. Movement: 4. Defense: 5. Regulation: 6. Transport: 7. Storage: 8. Stress Response: