1. Amino Acids and Proteins

2. 3-D Structure of Myoglobin

3. Importance of Proteins Main catalysts in biochemistry: enzymes (involved in virtually every biochemical reaction) Structural components of cells (both inside and outside of cells in tissues) Regulatory functions (if/when a cell divides, which genes are expressed, etc.) Carrier and transport functions (ions, small molecules)

4. Levels of Protein Structure Primary Structure - amino acid sequence in a polypeptide Secondary Structure - local spatial arrangement of a polypeptide’s backbone atoms (without regard to side chain conformation) Tertiary Structure - three-dimensional structure of entire polypeptide Quaternary Structure - spatial arrangement of subunits of proteins composed of multiple polypeptides (protein complexes)

6. Structure of  -amino acids

7. The 20 Amino Acids Found in Proteins

10. Properties of Different Amino Acid Side Chains

11. Stereochemistry of  -amino acids

12. Stereoisomers of  -amino acids All amino acids in proteins are L-amino acids, except for glycine, which is achiral.

13. RS Nomenclature System (Cahn, Ingold, Prelog System)

14. Leucine Cysteine All L-amino acids in proteins are S, except for cysteine, which is R. Alternative Representation of Amino Acids

15. Properties of Cysteine Side Chain Side chains with -SH or -OH can ionize, making them more nucleophilic. Oxidation between pair of cysteine side chains results in disulfide bond formation. Disulfide bonds are mainly found in extracellular proteins; the ~5 mM glutathione (  -Glu-Cys-Gly) makes the inside of the cell a highly reducing environment.

16. Hydroxyl-Containing Amino Acid Side Chains Serine Threonine Tyrosine

17. Tyrosine, Serine and Threonine Can Be Phosphorylated in Proteins Example: Tyrosine

18. Modified or Unusual Amino Acids

19. Absorption of UV Light by Aromatic Amino Acids

22. Titration of Amino Acids with Ionizing Side Chains Isoelectric point (pI) for amino acids with ionizable side chains: Take average pKa for the two ionizations involving the neutral (net charge of zero) species. pI of Glu = (2.19 + 4.25)/2 = 3.22 pI of His = (6.0 + 9.17)/2 = 7.59

23. Formation of a Peptide

24. Planarity of Peptide (Amide) Bond

25. .

26. cis and trans Isomers The trans isomer is generally more stable because of steric crowding of side chains in the cis isomer.

27. Examples of Oligopeptides

29. N- and C-Termini May Be Modified in Proteins

30. Primary Structure of Bovine Insulin First protein to be fully sequenced (by Fred Sanger in 1953). For this, he won his first Nobel Prize (his second was for the Sanger dideoxy method of DNA sequencing).

34. Evolution and Conservation of Protein Sequences Translation elongation factor Tu/1  Myoglobin

35. The Genetic Code

36. DNA RNA Protein

37. Initiating Amino Acid in Translation N-Formylmethionine in prokaryotes Just methionine in eukaryotes

38. Charging of tRNAs with Specific Amino Acids

39. Translation of mRNA into Protein

40. Ribosomal Peptidyl Transferase Activity Note: the catalytic component of the ribosome’s peptidyl transferase activity is RNA; it’s an example of a catalytic RNA or ribozyme.

41. Disulfide Bond Formation in Insulin

42. Methods in Protein Biochemistry

43. Gel Electrophoresis

44. Polyampholyte Character of a Tetrapeptide and Isoelectric Points Isoelectric Point (pI), pH at which molecule has net zero charge, determined using computer program for known sequence or empirically (by isoelectric focusing). Group pKa  -NH 3 + 9.7 Glu  -COOH 4.2 Lys  -NH 3 + 10.0  -COOH 2.2

46. Isoelectric Focusing Electrophoresis through polyacrylamide gel in which there is a pH gradient.

48. Two-Dimensional Gel Electrophoresis Separate proteins based on isolectric point in 1st dimension Separate proteins based on molecular weight in 2nd dimension

49. “Salting Out”: Ammonium Sulfate Precipitation in Protein Fractionation

50. Centrifugation Centrifugation Methods Differential (Pelletting) – simple method for pelleting large particles using fixed- angle rotor (pellet at bottom of tube vs. supernatant solution above) Zonal ultracentrifugation (e.g., sucrose- gradient) – swinging-bucket rotor Equilibrium-density gradient ultracentrifugation (e.g., CsCl) – swinging-bucket or fixed-angle rotor Low-speed, high-speed, or ultracentrifugation: different spin speeds and g forces

51. Zonal Centrifugation: Sucrose-Gradient Preparative Ultracentrifugation Separates by sedimentation coefficient (determined by size and shape of solutes)

52. Sucrose-Gradient Preparative Ultracentrifugation

53. Equilibrium Density Gradient Ultracentrifugation Used in Meselsen-Stahl experiment. Separates based on densities of solutes. Does not require premade gradient. Pour dense solution of rapidly diffusing substance in tube (usually CsCl). Density gradient forms during centrifugation (“self- generating gradient”). Solutes migrate according to their buoyant density (where density of solute = density of CsCl solution).

54. Column Chromatography Flow-through Eluate

55. Different Types of Chromatography Gel filtration/size exclusion/molecular sieve - separates by size (molecular weight) of proteins Ion exchange (cation exchange and anion exchange) - separates by surface charge on proteins –Cation exchange: separates based on positive charges of solutes/proteins, matrix is negatively charged –Anion exchange: separates based on negative charges of solutes/proteins, matrix is positively charged Hydrophobic interaction - separates by hydrophobicity of proteins Affinity - separates by some unique binding characteristic of protein of interest for affinity matrix in column

56. Ion-Exchange Chromatography

57. Gel Filtration Chromatography

58. Affinity Chromatography

59. Cleavage of Polypeptides for Analysis Strong acid (e.g., 6 M HCl) - not sequence specific Sequence-specific proteolytic enzymes (proteases) Sequence-specific chemical cleavage (e.g., cyanogen bromide cleavage at methionine residues)

60. Protease Specificities

61. Cyanogen Bromide Cleavage at Methionine Residues

63. Protein Sequencing: Edman Degradation PTH = phenylthiohydantion F 3 CCOOH = trifluoroacetic acid PTC = phenylthiocarbamyl

64. Identification of N-Terminal Residue Note: Identification of C-terminal residue done by hydrazinolysis (reaction with anhydrous hydrazine in presence of mildly acidic ion exchange resin) or with a C-terminus-specific exopeptidase (carboxypeptidase). NO 2

65. Separation of Amino Acids by HPLC

66. Protein Identification by Mass Spectrometry

67. Two main approaches: 1. Peptide mass fingerprinting: Proteolytic digestion of protein, then determination m/z of peptides by MS (e.g., MALDI-TOF or ESI-TOF), search “fingerprint” against database. Success of ID depends on quality/ completeness of database for specific proteome. 2. Tandem MS (MS/MS – e.g., nanoLC-ESI-MS/MS): Proteolytic digestion of protein, separation and determination of m/z of each (MS-1), then determination of collision-induced dissociation fragment spectrum for each peptide (MS-2). Gives context/sequence-dependent information, so more of a do novo sequencing method.

68. Locating Disulfide Bonds

69. Determing Primary Structure of an Entire Protein

70. Reactions in Solid-Phase Peptide Synthesis