1. Introduction to Molecular Biology zMolecular biology is interdisciplinary (biochemistry, genetics, cell biology) zImpact of genome projects (human, bacteria, fungi, plants, etc.)….”postgenomics era” zIntegration with other fields (e.g. computer science) leading to interdisciplinary career paths (Bioinformatics) Molecular Biology syllabus web siteweb site

2. Model Organisms

3. Central Dogma zDNA is transcribed to mRNA zmRNA is translated to protein

4. Proteins play many roles

5. Lecture 1 Protein Structure and Function Reading: Chapters 1-3

6. Protein Structure & Function -protein structure -protein purification & analysis -protein structure determination

7. Protein structure determines function zProteins are single, unbranched chains of amino acid monomers zThere are 20 different amino acids zA protein’s amino acid sequence determines its three-dimensional structure (conformation) zIn turn, a protein’s structure determines the function of that protein

8. Copyright (c) by W. H. Freeman and Company All amino acids have the same general structure but the side chain (R group) of each is different

9. Fig 3-2

10. Copyright (c) by W. H. Freeman and Company Hydrophilic amino acids Figure 2-13

11. Copyright (c) by W. H. Freeman and Company Hydrophobic & “special” amino acids Figure 2-13

12. Copyright (c) by W. H. Freeman and Company Peptide bonds connect amino acids into linear chains

13. Amino acids are the repeating units in proteins, but it is the 3-D protein structure that underlies function. How is 3-D structure obtained?

15. Copyright (c) by W. H. Freeman and Company Four levels of structure determine the shape of proteins zPrimary: the linear sequence of amino acids zSecondary: the localized organization of parts of a polypeptide chain (e.g., the  helix or  sheet) zTertiary: the overall, three-dimensional arrangement of the polypeptide chain zQuaternary: the association of two or more polypeptides into a multi-subunit complex

16. Secondary structure: the  helix Figure 3-3

17. Copyright (c) by W. H. Freeman and Company Secondary structure: the beta sheet

18. Copyright (c) by W. H. Freeman and Company Motifs are regular combinations of secondary structures A coiled coil motif is formed by two or more helices wound around one another

19. Copyright (c) by W. H. Freeman and Company Other examples of motifs

20. Copyright (c) by W. H. Freeman and Company Primary and secondary structure in hemagglutinin

21. Copyright (c) by W. H. Freeman and Company Tertiary and quaternary structure in hemagglutinin

22. Sequence homology suggests functional and evolutionary relationships between proteins Figure 3-10

23. Folding, modification, & degradation of proteins zA newly synthesized polypeptide chain must undergo folding and often chemical modification to generate the final protein zAll molecules of any protein species adopt a single conformation (the native state), which is the most stably folded form of the molecule

24. Copyright (c) by W. H. Freeman and Company The information for protein folding is encoded in the sequence

25. Copyright (c) by W. H. Freeman and Company Folding of proteins in vivo is promoted by chaperones Figure 3-15

26. Copyright (c) by W. H. Freeman and Company Chemical modifications and processing alter the biological activity of proteins

27. Copyright (c) by W. H. Freeman and Company Protein degradation via the ubiquitin-mediated pathway Figure 3-13 Cells contain several other pathways for protein degradation in addition to this pathway

28. Copyright (c) by W. H. Freeman and Company Aberrantly folded proteins are implicated is slowly developing diseases An amyloid plaque in Alzheimer’s disease is a tangle of protein filaments

29. Copyright (c) by W. H. Freeman and Company Functional design of proteins zProtein function generally involves conformational changes zProteins are designed to bind a range of molecules (ligands) yBinding is characterized by two properties: affinity and specificity zAntibodies exhibit precise ligand-binding specificity zEnzymes are highly efficient and specific catalysts yAn enzyme’s active site binds substrates and carries out catalysis

30. Copyright (c) by W. H. Freeman and Company Kinetics of an enzymatic reaction are described by V max and K m

31. Copyright (c) by W. H. Freeman and Company Mechanisms that regulate protein function zAllosteric transitions yRelease of catalytic subunits, active  inactive states, cooperative binding of ligands zPhosphorylation  dephosphorylation zProteolytic activation zCompartmentalization

32. Copyright (c) by W. H. Freeman and Company Purifying, detecting, and characterizing proteins zA protein must be purified to determine its structure and mechanism of action zMolecules, including proteins, can be separated from other molecules based on differences in physical and chemical properties

33. Copyright (c) by W. H. Freeman and Company Integral membrane proteins can be solubilized by non-ionic detergents

34. Copyright (c) by W. H. Freeman and Company Centrifugation can separate molecules that differ in mass or density

35. Copyright (c) by W. H. Freeman and Company Electrophoresis separates molecules according to their charge:mass ratio SDS-polyacrylamide gel electrophoresis

36. Copyright (c) by W. H. Freeman and Company Two-dimensional electrophoresis separates molecules according to their charge and their mass

37. Copyright (c) by W. H. Freeman and Company Separation of proteins by size: gel filtration chromatography

38. Copyright (c) by W. H. Freeman and Company Separation of proteins by charge: ion exchange chromatography

39. Copyright (c) by W. H. Freeman and Company Separation of proteins by specific binding to another molecule: affinity chromatography

40. Copyright (c) by W. H. Freeman and Company Highly specific enzymes and antibody assays can detect individual proteins

41. Copyright (c) by W. H. Freeman and Company Protein primary structure can be determined by chemical methods and from gene sequences Edman degradation

42. Copyright (c) by W. H. Freeman and Company Time-of-flight mass spectrometry measures the mass of proteins and peptides

43. Copyright (c) by W. H. Freeman and Company X-ray crystallography is used to determine protein structure Other techniques such as cryoelectron microscopy and NMR spectroscopy may be used to solve the structures of certain types of proteins