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Chapter 5

Chapter 5. Introduction to Studying Proteins. Learning Objectives. Describe the structure of proteins Explain transcription and translation Discuss naturally occurring and recombinant proteins in biotechnology Learn the function of proteins in structure, recognition and as catalysts

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Chapter 5

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  1. Chapter 5 Introduction to Studying Proteins

  2. Learning Objectives • Describe the structure of proteins • Explain transcription and translation • Discuss naturally occurring and recombinant proteins in biotechnology • Learn the function of proteins in structure, recognition and as catalysts • Explore enzymes and conditions of activity • Understand PAGE

  3. Structure and Function of Proteins • Proteins are essential parts of organisms and participate in virtually every process within cells • Proteins make up half the dry weight of an Escherichia colicell, whereas other macromolecules such as DNA and RNA make up only 3% and 20%, respectively

  4. Structure and Function of Proteins • Biotech products are often proteins or related to proteins • Human growth hormone • BEANO (a-galactosidase) • Monosodium glutamate • Amylase • ELISA • Vaccines • Toxins (black widow, cholera)

  5. Structure and Function of Proteins Proteins carry out the duties specified by the information encoded in genes The set of proteins expressed in a particular cell or cell type is known as its proteome

  6. Structure and Function of Proteins Characteristic Measured by Mass spectrometer ionizing chemical compounds to generate charged molecules sorts the ions by mass by applying electromagnetic field • Molecular mass • Normally reported in units of daltons (synonymous with atomic mass units), or the derivative unit kilodalton (kDa). • Insulin 4kDa in mass • Muscle protein (myosin)250kD in mass

  7. Structure and Function of Proteins Characteristic Three-Dimensional Structure Methods X-ray Crystallography Pure crystals of proteins are generated X-ray are directed on the crystals Position of atoms diffract the x-ray Diffraction pattern analyzed by computer programs to generate a 3-D picture

  8. Structure and Function of Protein Chemical behavior Activity Catalysis Structure Movement Defense Regulation Transport

  9. Structure and Function Solubility Dependant on Shape Fibrous proteins with long, rod-shaped molecules Globular proteins are compact spherical molecules that usually bind other molecules • Insoluble in water—keratins found in skin, hair and nails, • Soluble in water—nearly all enzymes and immunoglobulin's—dynamic functions

  10. Structure and Function of Proteins Charge Electrophoresis/Chromatography Separates on charge and size PAGE = Polyacrylamide gel electrophoresis Use SDS to impart a negative charge on all proteins • Charge is determined by R groups on the amino acids in the protein

  11. To Summarize:The structure and Function of Proteins • Mass • Three dimensional structure • Activity • Solubility • Charge

  12. Protein Molecule Structure • Proteins are polymers of amino acids • Common structure • Central Carbon atom • Amino Group • Carboxylic acid • R-group • H H H2N – C--COOH R

  13. Amino Acids • Twenty different amino acids • Categorized by the nature of the R group • Zwitterions (Carry both a positive and negative charge in water solutions) H O H2N – C--COH R

  14. Amino Acids • Twenty different amino acids • Categorized by the nature of the R group • Zwitterions (Carry both a positive and negative charge in water solutions) H O +H3N – C—CO- R

  15. Amino Acids and Polarity • The interaction of amino acids with a water solution (the cytosol) and with other amino acids in a protein will depend upon the polarity of the R group.

  16. Polar water molecule • Unequal sharing of electrons in a covalent bond leads to a polar molecule. • IN WATER: • The region around oxygen bears a partial negative charge • The region around the hydrogens bear partial positive charge • Water forms hydrogen bonds

  17. What does it mean to be polar? Oil and Water do not mix • Very different bonding characteristics • Must have bonding similar to water to interact with water • These interactions drive the secondary and tertiary structure of proteins

  18. Amino Acids • R groups may be: • Acidic…………………………………..Loss of a proton (H+) Negatively charged • Basic…………………………………….Gain of a proton (H+) Positively charged • Polar……………………………………..Characterized by OH Interact with water and other polar groups • Hydrophobic………………………….Characterized by C-H • Repelled by water

  19. Amino acids Acidic Amino Acid Basic

  20. Amino Acids Nonpolar Polar

  21. Primary StructureThe sequence of amino acids in the protein

  22. Secondary Structure :Hydrogen bonding between atoms in the peptide bond can produce regular repeating patterns Beta- Pleated Sheet Alpha Helix

  23. Secondary Structure

  24. GFP-Structural Barrel with Beta -Pleated Sheet

  25. Tertiary Structure: Overall folded pattern of a Globular Protein

  26. Sickle-Cell anemia due to change in molecular shape • An A to T mutation of the β-globin gene results in the amino acid glutamate to be substituted by valine at position 6 of the polypeptide • The genetic disorder is due to the mutation of a single nucleotide, from a GAG to GTG codon mutation.

  27. When deoxygenated in the capillaries, the S-hemoglobin has a hydrophobic patch on the protein • The hydrophobic residues of the valine at position 6 of the beta chain in hemoglobin are able to associate with the hydrophobic patch, causing hemoglobin S molecules to aggregate and form fibrous precipitates

  28. Changes to primary structure lead to changes in tertiary structure and function • HbS molecules tend to clump together, making red blood cells sticky, stiff, and more fragile, and causing them to form into a curved, sickle shape.

  29. Quaternary Structure: 2 or more subunits associate to form the functional protein Hemoglobin DNA Polymerase

  30. http://wpcontent.answers.com/wikipedia/commons/b/ba/Hemoglobin_t-r_state_ani.gifhttp://wpcontent.answers.com/wikipedia/commons/b/ba/Hemoglobin_t-r_state_ani.gif

  31. Proteins grouped by function • Structural • Enzyme • Transport • Contractile • Hormone • Pigment • Recognition • Toxins • Antibody • Collagen, keratin • Amylase, rennin • Hemoglobin, HDL • Actin, myosin, tubulin • Insulin, Adrenalin • Melainin, rhodopsin • Glycoprotein, MHC • Botox, tetanus • Gamma globulin, IgE

  32. Antibodies: Structure and Function RECOGNIZE, BIND & NEUTRALIZE STRUCTURE CRITICAL TO FUNCTION Thousands of different foreign invaders need to be recognized—NEED VARIETY All antigens will be recognized, bound, marked and clumped together for removal – NEED A CONSTANT REGION • Recognize foreign proteins called antigens • Tag and aggregate antigens • Remove or neutralize antigenic proteins in the body • Molecular weapons of defense

  33. Each Antibody has Same Basic Shape • Y-shaped glycoprotein protein • Made of 4 polypeptide chains • Two heavy chains • Two light chains

  34. Antibody structure • Variable region—identifies all the many possible antigens • Constant region that is identical for all antibodies –the amino acid sequence is the same for each class of antibodies • Held together by disulfide bonds

  35. Types of Antibodies • There are several different types of antibody heavy chains, and several different kinds of antibodies, • Grouped into different isotypes based on which heavy chain they possess. • Five different antibody isotypes are known in mammals • Perform different roles • Direct the appropriate immune response for each different type of foreign object they encounter.

  36. Five Antibody Isotypes • Five antibody isotypes known as IgA, IgD, IgE, IgG and IgM. • They are each named with an "Ig" prefix that stands for immunoglobulin (another name for antibody) • Differ in their biological properties, functional locations and ability to deal with different antigens

  37. The basic structural units—each with two large heavy chains and two small light chains. • May form monomers with one unit, dimers with two units or pentamers with five units

  38. Variable region allows for recogniton of various antigens • The small region at the tip of the protein is extremely variable • Known as the hypervariable region. • Each will bind to a different antigen.

  39. Diversity of antibodies allows the immune system to recognize a diversity of antigens • The unique part is called an epitope. • A highly specific interaction with antigen by induced fit • Recognition of an antigen by an antibody tags it for attack by other parts of the immune system. • Neutralize targets directly by binding to a part of a pathogen that it needs to cause an infection.

  40. Genetic Shuffle Creates Variety • The diversity of antibodies is due to random combinations of a set of gene segments • Gene encodes different antigen binding sites • Random mutations in this area of the antibody gene to create further diversity. • Antibody genes also re-organize in a process to change the base of the heavy chain to another

  41. Antibodies (AB) Mark Antigens (AG)for Elimination • Binding of AB to AG • Neutralize the AG • Agglutinate microbe • Precipitate dissolved antigen • Activate complement • Enhances phagocytosis • Leads to cell lysis

  42. Antibody Variability • Creates a different isotype of the antibody but retains the antigen specific variable region • Allows a single antibody to be used by several different parts of the immune system

  43. Problems with Immune System • Autoimmune disorders • Antibodies against self • (Lupus, R.Arthritis, MS, Diabetes) • Immunodeficiency disease • (SCID, Hodfkin’s, AIDS) • Allergies • Abnormal Sensitivity to antigens

  44. Allergens • Antigens that induce the formation of IgE antibodies • Excess IgE stimulates an inflammatory response • Allergen binds to antibodies bound to MAST cells • Mast cells release histamine • Blood vessels dilate and leak fliud • Nasal irritation, itchy skin and tears • Aanaphylactic shock—so rapid– blood pressure falls

  45. Ouchterlony Test for antigen-antibody • Hundreds of AB-AG complexes form to produce a precipitation

  46. Poison Ivy Exaggerated Immune Response • The poison ivy plant and its relatives are common throughout the United States. • Poison ivy leaves are coated with a mixture of chemicals called urushiol

  47. Allergic Contact Dermatitis Allergy is an altered or unwanted immune response Dermatitis is an inflammation of the skin Response to something which came into contact with the skin Poison ivy, other things which contact the skin such as clothing, shampoo, jewelry, make-up, and deodorants can also cause allergic contact dermatitis. Also can be caused from something we ate. • The immune system neutralizes and eliminates foreign substances from our bodies. • It sometimes attacks harmless substances vigorously, causing an inflammation which can be far more harmful than the foreign substance alone.

  48. Toxic Effects of Urushiol are indirect, mediated by an induced autoimmune response Urushiol acts as a hapten, • chemically reacting with • binding to and • changing the shape of integral membrane proteins on exposed skin cells. Affected proteins no longer are recognized as normal parts of the body, causing an immune response

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