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Protein Structure and Function

Protein Structure and Function. ChE 170 Lecture 10/18/11. Protein Function is Dictated by its Structure. Enzyme activity can depend on structural conformation Unique binding sites in antibodies dictate the specific ligand to which the antibody binds. Active State.

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Protein Structure and Function

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  1. Protein Structure and Function ChE 170 Lecture 10/18/11

  2. Protein Function is Dictated by its Structure • Enzyme activity can depend on structural conformation • Unique binding sites in antibodies dictate the specific ligand to which the antibody binds Active State Pinkas et al PLoS Biol (2007)

  3. Herceptin and HER2 PDB ID: 1N8Z Cho, H.-S. et al. Nature (2003).

  4. So Why Do We Care about Protein Structure/Function? Discussion

  5. Importance of Understanding Protein Binding Interactions • Antibodies and the immune response • Binding of agonistic ligands to cell surface receptors • GPCR’s such as the AT1 receptor • Engineering therapeutic drugs • Specificity and side effects • Affinity affects transport • Wittrup’s model Thurber, Schmidt, & Wittrup. Trends in Pharmacological Sciences (2007).

  6. What Mediates Protein Structure? • Destabilizing conditions • Heat • Chemicals (urea) • Extreme pH • High salt concentrations • Reducing agents • Can be reversible

  7. What Mediates Protein Structure? • Protein folding occurs on the ribosome • Chaperones • Further processing involves additional enzymes • Isomerase • Disulfide bond formation • Protease activation • Protein misfolding • Ubiquitin • Can lead to disease H3N+ COO- kf H3N+ COO-

  8. Important Secondary Structures • Alpha-helix • Cylindrical structure: hydrogen bonded backbone • Residue n h-bond with n+4 • Beta-sheet • Network of hydrogen bonds: antiparallel vs. parallel Beta-Barrel PDB: 1EMA

  9. Stabilizing Forces Covalent Bond Disulfide Bond Salt Bridge Hydrogen Bond Long-range Electrostatic Interaction Van der Waals Interaction Petsko & Ringe; Protein Structure and Function; New Science Press; 2004; pg. 11

  10. Protein Binding Interaction

  11. Binding Affinity Dictates Half-Life

  12. Protein Dissociation Example

  13. Use Surface Plasmon Resonance to Determine Binding Affinity

  14. Surface Plasmon Resonance

  15. Thermodynamics of the Binding Interaction

  16. Antibodies: Structure and Function

  17. Antibodies: Natural Functions • Several classes of immunoglobulins • IgG, IgA, IgM, IgD, IgE (arranged by half-life high to low) • IgG are the most abundant Nester, Anderson, Roberts, and Nester; Microbiology: A Human Perspective; McGraw Hill; 2007; pg 394

  18. Antibodies: Engineered Uses • In vitro diagnostics • ELISA’s • Largest class of biologic therapeutics • Important for research in biology and medicine • Human Protein Atlas

  19. Figure 3-1 part 1 of 3

  20. IgGs are Composed of Two Types of Protein Chains

  21. IgGs have Two Important Domains

  22. Figure 3-1 part 2 of 3

  23. Figure 3-3

  24. Anti-parallel Beta-Sheets

  25. Figure 3-5 part 1 of 2

  26. Figure 3-5 part 2 of 2

  27. The Hypervariable Regions

  28. Figure 3-7

  29. Figure 3-8 Antibodies Bind in Different Ways

  30. How Do We Generate Antibodies for our Own Purposes? • Polyclonal Mixtures • Animal immunizations limited supply • Heterogeneous binding specificities • Significant need to generate monoclonal antibodies Hybridomas! • B-cells (produce IgG) fused with myeloma cells to produce hybrid myelomas that secrete IgG and grow continuously

  31. Production of Monoclonal Antibodies from Hybridoma Cells

  32. Production of Monoclonal Antibodies from Hybridoma Cells Fluorescence-Activated Cell Sorting (FACS)

  33. PMT 90º Light-Scatter (SSC) Non-target cells Target cells Dichroic mirror Red fluorescence Piezo Band-pass filters Green fluorescence LASER Forward light-scatter (FSC) Cell sample Fluorescence-Activated Cell Sorting (FACS)

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