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 Pinkas et al PLoS Biol (2007) Active State

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

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 AT 1 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+H3N+ COO - kfkf H3N+H3N+

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 KDKD t 1/2 Example mMmsNon-specific μMμMms - s Multivalent cell surface; intracellular signaling proteins nMmin-hrAntibodies pMhr- daysGrowth factors/receptor fMweeks-monthsStreptavidin-biotin

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

23. Figure 3-1 part 2 of 3

24. Figure 3-3

25. Anti-parallel Beta-Sheets

26. Figure 3-5 part 1 of 2

27. Figure 3-5 part 2 of 2

28. The Hypervariable Regions

29. Figure 3-7

30. Figure 3-8 Antibodies Bind in Different Ways

31. 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

32. Production of Monoclonal Antibodies from Hybridoma Cells

33. Fluorescence-Activated Cell Sorting (FACS)

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