Week 5 Overview Of Protein Function - Biochemistry PDF

Week 5 Overview Chapter 5; Protein Function For Tuesday: – Ligand Binding, Myoglobin and Hemoglobin For Thursday – Structural Proteins, Motor Proteins and Antibodies Other – Wiley Plus Practice Problems – Weekly Problem Set #3. – For Thurs: Read Paper (SCD vs CFTR funding) an...

Week 5 Overview Chapter 5; Protein Function For Tuesday: – Ligand Binding, Myoglobin and Hemoglobin For Thursday – Structural Proteins, Motor Proteins and Antibodies Other – Wiley Plus Practice Problems – Weekly Problem Set #3. – For Thurs: Read Paper (SCD vs CFTR funding) and post reflection question answers – Topic Selectin for Biochemistry and Society Project Key Concept: The structure of a protein enables its function 5.2. Structural Proteins provide support both within and outside the cell.  Two categories of structural proteins Globular proteins that associate – Actin – microfilaments – Tubulin – microtubules Fibrous proteins that are elongated – Keratin – intermediate fibers – Collagen (extracellular protein)  F-actin is a polymer of the monomeric G actin protein Polymerization Globular protein (~375 aa) with ATP binding site. Actin Filament Assembly (F-actin) Polymerization driven by ATP hydrolysis Copyright © 2017 John Wiley & Sons, Inc. All rights reserved.  Functions of Actin filaments Cell shape/ size/ structural support Cell movement Muscle movement Copyright © 2017 John Wiley & Sons, Inc. All rights reserved. Microtubules are polymers of the tubulin dimer Globular proteins (~450 aa) with GTP binding site. Copyright © 2017 John Wiley & Sons, Inc. All rights reserved. Actin filaments vs Microtubules; structure vs flexibility Copyright © 2017 John Wiley & Sons, Inc. All rights reserved. Microtubules are hollow fibers built from tubulin dimers 13 filaments per tube. GTP hydrolysis occurs upon polymerization  Microtubule Functions Cytoskeleton – support Cell division Cell movement (cilia, flagella) Transport within cell Microtubules Chromosomes  Fibrous proteins provide structural support. Fibrous proteins are characterized by well defined secondary structure. 𝛂- keratin Collagen  Keratins are a class of structural proteins Backbone Stick Space- filling α-Keratin has coiled-coil structure. Large hydrophobic residues repeat every 4 positions. How does this feature impact the protein structure? What does high level of Cys indicate? Assembly of filament Disulfide bonds can form between fibers to stabilize Collagen is part of the Extracellular Matrix Copyright © 2017 John Wiley & Sons, Inc. All rights reserved.  Collagen fibers formed from tropocollagen; a triple helix protein  Contains high levels of Glycine and proline Collagen has a unique amino acid composition; Gly-X-Y triple motif Every 3rd amino acid is glycine and 30% of remaining are Pro or Hyp Hyp is formed via a reaction requiring vitamin C. Cross-section of the Collagen Triple Helix; Gly align in center Polypeptides are crosslinked Collagen fiber is assembled from multiple tropocollagen proteins  Review Key Concepts 1. Two categories of structural proteins are ______ proteins that associate and ______ that are elongated. 2. Actin filaments are composed of ______ while microtubules are composed of ______. Of these two, ____ are more flexible than ______. 3. ___ are important in muscle movement while ____ are important in cell division. 4. Fibrous proteins are have well defined _____ structure. 5. ____ is a dimer of alpha helices that wind around each other while ____ is a trimer that forms a triple helix. 6. ______ is found in the extracellular matrix of cells.  5.3. Motor proteins: facilitate movement by acting on structural elements. Convert chemical energy to mechanical energy. Myosin has two heads and a long tail The heads have ATP and actin binding sites, and a flexible neck. ATP hydrolysis drives the physical movement of myosin along an actin filament Kinesin is a microtubule-associated protein. Opposite end binds to a Heads contain nucleotide protein in a vesicle. binding site and tubulin binding site.  Kinesin transports cargo by moving processively along a microtubule track. Copyright © 2017 John Wiley & Sons, Inc. All rights reserved. Defensive Protein: Antibody Structure and Function Antibodies recognize and bind to “foreign” molecules Large proteins with huge amino acid variation Important tool used in biochemistry to identify specific proteins.  Immune response activated by foreign substances. Antigen = foreign substance Antibody = immune protein that binds it. Epitope (antigenic determinant) region where antibody binds.  Antibodies are produced by immune cells Immune cells produce surface antibodies. Upon exposure to antigen soluble antibodies are produced. Make a prediction about how the structure of the surface antibodies differs from those of the soluble antibodies. Overview of simplified immune response Memory cells are also produced upon initial exposure to antigen. Allow more rapid immune response upon second exposure. How does this relate to the development of vaccines?  The Structure of Antibodies Schematic model of an IgG antibody 4 polypeptide chains – 2 “heavy chains” ~53 kDa – 2 “light chains” ~ 23kDa Chains held together by disulfide bonds C = constant domains V= variable domains The Antigen-Binding Site is hypervariable and comprises the complementarity- determining regions of the sequence. Shape and charge complementarity allow for specific interaction between antibody and antigen. Antibody and viral peptide showing shape complementarity  Antibodies are used as biochemical tools to identify, quantify, or localize particular proteins ELISA Immunoblotting Immunocytochemistry How are antibodies made? Indirect enzyme-linked immunosorbent assay (ELISA), test for presence of protein Immunoblotting (Western Blotting) used to identify / quantify proteins in a complex mixture Similar to ELISA Proteins separated on gel & then transferred to membrane. Membrane probed with antibody Secondary antibody used to visualize primary antibody  Immunocytochemistry allows for localization of particular proteins in cells/tissues mixture Antibodies with fluorescence tags are used Localization using microscopy  Key Learning Outcomes What should you be able to do after learning this material? Be able to compare the structures and functions of structural proteins How do the globular proteins that assemble differ from the fibrous proteins and how does that impact their structural roles? How do structural features, such as amino acid sequences, ability to cross link, impact the overall structure and therefore function of these proteins? Explain how motor protein operate (in general) Describe the general structure of motor proteins Describe the structure and function of immunoglobulins (antibodies) Identify the key features of antibodies Be able to describe the applications of antibodies in the laboratory and clinic.  Review Key Concepts 1. The ______ of a protein allows for its ______. 2. An ______ is a foreign substance that an _____ binds to during an immune response. 3. An antibody contains ___ heavy chains and __ light chains that held together by ______. 4. The complementarity of both ____ and ____ allow for the specificity of the interaction between antiboides and antigens. 5. The ____ allows for testing for the presence of a protein while the technique of ____ is used to identify and/or quantify proteins in mixtures. 6. _______ is used to figure out the location of proteins within cells.