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Protein Function –Binding

Protein Function –Binding. Chapter 5:. Function of Globular Proteins: Ligand Binding. Learning Goals. 1. Reversible binding of ligands is essential Specificity of ligands and binding sites Ligand binding is often coupled to conformational changes, sometimes quite dramatic (Induced Fit)

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Protein Function –Binding

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  1. Protein Function –Binding Chapter 5:

  2. Function of Globular Proteins: Ligand Binding Learning Goals 1. Reversible binding of ligands is essential • Specificity of ligands and binding sites • Ligand binding is often coupled to conformational changes, sometimes quite dramatic (Induced Fit) • In multisubunit proteins, conformational changes in one subunit can affect the others (Cooperativity) • Interactions can be regulated 2. Illustrated by: • Hemoglobin, antibodies, and muscle contraction

  3. Functions of Globular Proteins • Storage of ions and molecules • myoglobin, ferritin • Transport of ions and molecules • hemoglobin, serotonin transporter • Defense against pathogens • antibodies, cytokines • Muscle contraction • actin, myosin • Biological catalysis • chymotrypsin, lysozyme

  4. Protein Interaction with Other Molecules • Reversible, transient process of chemical equilibrium: A + B AB • A molecule that binds to a protein is called a ligand • Typically a small molecule • A region in the protein where the ligand binds is called the binding site • Ligand binds via same noncovalent forces that dictate protein structure (see Chapter 4) • Allows the interactions to be transient

  5. Hemoglobin and Oxygen/CO2 Binding

  6. Structures of Porphyrin and Heme

  7. Coordination Positions – Bind with His93 = His F8

  8. Myoglobin

  9. Ligand Binding θ = Fraction of Protein’s Ligand Binding Sites Bound to Ligand

  10. Oxygen Binding to Myoglobin

  11. What are the Kd’s ? A EOC Problem 1: Relates Kd with “affinity” for ligand

  12. How is the Binding Experiment Done? With proteins such as Hemoglobin and Myoglobin, the absorbance spectrum changes between free and bound protein can be measured in a spectrophotometer. The spectrum of deoxy-myoglobin is different than oxy-myoglobin. What about proteins without a chromophore? and binding colorless ligands? Equilibrium dialysis

  13. Human serum transferrin binds iron at pH 7.2 with a Kd of 10-19 to 10-20 M EOC Problem 5 uses simple inspection of the data to get Kd ! Make sure you get this done before Class.

  14. Carbon Monoxide Binds Heme better than Oxygen Due to Steric Effect of His E7 (see next slide)

  15. Comparison of Myoglobin and a Hemoglobin Polypeptide

  16. Amino Acid Sequence of Myoglobin and Hemoglobin Polypeptides Grey Conserved, Pink Conserved in all known Hemoglobins

  17. Hemoglobin Structure Showing Interchain Contact Points

  18. Contact Points in Primary Structure

  19. Hemoglobin has Two Different Folded States

  20. T  R

  21. Oxygen Binding Curves EOC Problem 6 gets you further into cooperativity in oxygen binding. Knowing this will help in Class.

  22. Cooperative Binding

  23. Hill Plot

  24. CO Binds Well Increased Exposure to even Low Levels of CO results in COHb! + The effect of exercise is trivial.

  25. This is Fatal

  26. Two Models of Sigmoid Curves Monod-Wyman-Changeux Koshland (Induced Fit)

  27. Both Models Fit the Data

  28. Hb Oxygen Carrying Ability: the Bohr Effect

  29. CO2 Transport on N-terminal Amino Groups

  30. Effect of Altitude and 2,3 bisphosphoglycerate EOC Problem 3: examines this phenomena...be sure you know this for Class.

  31. BPG Fits into the Hole of the Doughnut R state without and with BPG Positively charged R groups are in blue.

  32. Normal RBC vs Sickle Cell RBC’s

  33. The Difference In Shape is due to One Amino Acid Change Glu6 Val6 in Beta

  34. Separation of Protein FragmentsThe Classic Paper Chromatography + Electrophoresis Image from Chapter 3

  35. Hb-S Polymerizes in the Deoxy form

  36. Lymphocytes Have Binding Proteins A

  37. Antibodies have at least 2 Antigen Binding Sites IgG Carbohydrate Bound Here

  38. IgG Protein Structure and Binding an Antigen

  39. IgM Has 10 Ag Binding Sites IgG and IgM are the major circulating antibodies

  40. Cartoon of an ELISA

  41. ELISA to Detect Herpes Simplex Virus in Blood Samples Positive Negative Controls

  42. Antibodies Used with PAGE: Immunoblots

  43. Binding receptors initiates Phagocytosis. A

  44. Myosin

  45. Myosin Aggregate

  46. Actin Filament

  47. Myosin Contacting an Actin Filament

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