BIOL 302 1st Edition Lecture 18Outline of Last Lecture I. Covalent modificationII. Membrane StructureIII. Membrane transport: Membrane channels, carriers and pumpsOutline of Current Lecture I. AntibodiesCurrent LectureI. Clicker question: Which of the following is a catalytic mechanism that enzymes use:A. Stabilization of the productB. Stabilization of the substrateC. Solvation of the bound substrateD. Decreasing the entropy of bound substrateE. Favorable electrostatic interactions at the substrate binding siteII. Clicker question: In ATCase, the substrate CTP is A. a heterotropic allosteric inhibitorB. a homotropic allosteric inhibitorC. a competitive inhibitorD. a non-competitive inhibitorE. a non-allosteric inhibitorIII. Clickerquestion: Which signal targets proteins for degradation? A. PhosphorylationB. SUMOylationC. FarnesylationD. UbiquitinylationE. All of the above.IV. Clicker question: Which of the following is not true about transmembrane proteins? These notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.A. Transmembrane proteins can be anchored in the membrane by various ‘anchors’ such as GPI (Glycosylphosphatidylinositol).B. Transmembrane α-helices are the most common structural elements of membrane proteins. C. β-barrels can span membranes.D. Charged residues are mainly in the soluble domains.E. The membrane-aquous boundary is often marked by tryptophan residues.V. Clicker question: Which of the following is not true about phospholipids? A. Phospholipids form micelles.B. Phospholipids are made in the endoplasmatic reticulum. C. Sphingomyelin is a phospholipid.D. Phospholipids consist of a glycerol substituted with a fatty acid and a phosphate group with a hydrophilic head group.E. Some phospholipids have specific binding sites on membrane proteins and have specific physiological roles. VI. Clicker question: Which of the following is true about phospholipids? A. Phospholipids associate with lipid rafts. B. Phospholipids can be used to solubilize membrane proteins.C. Phospholipids always have a net negative charge.D. A membrane with phospholipids is thicker than a membrane with Cholesterol.E. Phospholipids can diffuse laterally through the membrane.VII. Clicker question: What is the main type of interaction between this antigen and the residues on the antigen-binding site?A. Electrostatic forcesB. Hydrogen bondinC. Hydrophobic forcesD. Disulfide bondingE. Covalent bondsVIII. Chapter 3.3, & 34A. Antibodies:Origin, structure and applicationIX. Origin of AntibodiesA. Produced by B-cells in response to a foreign substance, called an antigenB. Immune system can produce more than 108 distinct antibodiesX. Antigen presentationA. B-cell activation requires presentation of antigenic peptides on a Class II major histocompatibility complex (MHC)B. T-helper cell that can recognize portions of the MHC/peptide complex promote growth and differentiation of the B-cell. XI. B-cell activationA. Only those B cells that can bind Ag and have specific activated T-helper cell will proliferate, forming memory cells. A few B cells differentiate into plasma cells that produce and secrete antibodiesXII. Kinetics of antibody productionA. Subsequent immune response is faster & stronger.XIII. Membrane vs Secreted AbA. B cells express Ig Heavy chain transcripts that include transmembrane and cytoplasmic domainsB. Plasma cells express Ig Heavy chain transcripts that stop after the CH domains, thereby encoding the same antibody but in a secreted formXIV. Antibody StructureA. Antibodies are Y-shaped structure containing four polypeptide chains. B. Antibodies have two identical light chains (pink), and two identical heavy chains (blue), which are at least twice as long as light chains.XV. Antibody-Antigen complexXVI. Antibody/Antigen binding is very strong and very specificA. Antigen/Antibody binding involves non-covalent interactions and can be very strong, with dissociation constants in the range of 10-7 to -10 MXVII. Antibody binding small molecule antigenA. Shape fitB. Non-covalent forces of interactionXVIII. Antibodies can be polyclonal or monoclonalA. Polyclonal antibodies1. Immunize with antigenAntibodies produced “natural way”2. Multiple epitopes if antigen is large3. Multiple B cell clones4. Multiple types of antibodies with diversity inepitope recognizedXIX. Polyclonal antibodiesA. How could we enrich the amount of antibody that recognizes our antigen?1. Gel filtration2. Crystallization3. Affinity purification 4. Ion exchange purification5. All of the aboveXX. Monoclonal AntibodiesA. Antibodies from single clone of B cellsB. Binding at only one epitopeC. Homogeneous in antigen binding characteristicsD. ReproducibleXXI. Making a monoclonalXXII. Alternative to monoclonal antibodies:A. Nanobodies, natural single domain antibodies1. A Llama is immunized with the antigen 2. Blood serum is collected 3. All antibodies are sequenced3. A plasmid library of all antibody DNA sequences is made4. Antibodies are recombinantly expressed in E. coli and purified 5. Antibodies are characterizedXXIII. Antibodies are powerful detection reagentsbased on their selective binding to antigen (Ag)A. To detect presence of AgB. To measure concentration of AgC. To purify the AgD. To localize a Ag in cells and tissuesXXIV. Antibodies as toolsA. Western Blot1. Directly conjugating to a reporter that enables detection such asa. enzyme (western blots) b. --gold particle (EM), c. --fluorophore (confocal microscopy),XXV. Antibodies as toolsA. Western Blot1. Separate proteins according to their electrophoretic mobility (a function of length of polypeptide chain or molecular weight). 2. Electrophorectically transfer to a membrane (blot)3. Incubate blot with Antibody4. Location of the protein/antibody complex is revealed by incubating the blot with a colorless substrate that the attached enzyme (AP) converts toa colored product that can be visualized and photographed.XXVI. Visualizing antigen/antibody complexA. Antibody conjugated to enzyme HRP = horseradishperoxidaseB. Substrate reacts to form chemiluminescent productC. Expose blot to film to measure chemiluminescenceD. Scan to quantify stain intensityXXVII. Antibodies as clinical toolsA. ELISA - Can test multiple samples for presence of specific protein,Can also perform quantitative assaysXXVIII. ELISA for antibodies, or for antigenA.