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

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

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

12. Fig 3-2

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?

14. 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

15. Secondary structure: the  helix Figure 3-4 The spiral is held by hydrogen bonds between nearly adjucent backbone O and H atoms

16. Copyright (c) by W. H. Freeman and Company Secondary structure: the beta sheet Hydrogen bonds occur between backbone O and H of separate ajucent strands

17. 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

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

19. Tertiary structure // quaternary structure hemagglutinin Regions of proteins form domains: functional, topological or structural (like in case of HA) Hydrophobic, hydrophylic interactions and disulfide bonds help to keep the structure The structure is stabilized by Interactions between domains

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

21. 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

22. The information for protein folding is encoded in the sequence

23. Folding of proteins in vivo is promoted by chaperones

24. 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

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

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

27. 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

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

29. 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

30. 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

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

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

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

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

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

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

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

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

39. 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