1. Biochemistry Sixth Edition Chapter 3: Exploring Proteins and Proteomes Copyright © 2007 by W. H. Freeman and Company Berg Tymoczko Stryer

4. The proteome is the functional representation of the genome Proteome: proteins expressed by the genome Represents functional expression of information Larger than the genome

5. 3.1 The purification of proteins is an essential first step In understanding their function Never waste pure thoughts on an impure protein

6. The assay: how do we recognize the protein that we are looking for? Assay: a test for some unique identifying property of the protein Specific activity: the ratio of enzyme activity to the amount of protein Purification: to maximize the specific activity

7. An assay for LDH

8. Proteins must be released from the cell to be purified Fractionation Homogenate Differential centrifugation

10. Proteins can be purified according to solubility, size, charge, and binding affinity Salting out with ammonium sulfate Dialysis Gel-filtration chromatography Ion-exchange chromatography Affinity chromatography High-pressure liquid chromatography: high resolution, rapid separation

11. Dialysis

12. Gel-filtration chromatography

13. Ion-exchange chromatography

15. Affinity chromatography

16. HPLC

17. Proteins can be separated by gel electrophoresis and displayed Gel electrophoresis v = Ez /f, f = 6  r The gel serves as a molecular sieve SDS-PAGE: separation on the basis of mass under denaturing condition One SDS for every two amino acid residues Mercaptoethanol: reducing agent Staining

18. PAGE

21. Formation of a polyacryamide gel

22. SDS

23. staining

24. Mass vs. mobility

25. Isoelectric focusing Isoelectric point Generation of pH gradient, polyampholytes Two-dimensional gel electrophoresis Combination of isoelecrtic focusing and SDS-PAGE

26. IEF

27. 2D gel elctrophoresis

28. E. coli proteins

29. A protein purification scheme can be quantitatively evaluated Total protein Total activity Specific activity Yield Purification level

31. Electrophoretic analysis of purification

32. Ultracentrifugation is valuable for separating biomolecules and determining their masses s = m(1-  f  partial specific volume  density of the medium Sedimentation coefficient x   r = velocity Svedberg unit (S): 10 -13 s Sedimentation velocity depends on mass, shape, density, and density of the medium Gradient centrifugation

34. Density and sedimentation coefficients

37. Gradient centrifugation

39. 3.2 Amino acid sequence can be determined by automated Edman degradation Sequencing a peptide Amino acid composition – heating in 6N HCl at 110 C for 24 hrs Identification using HPLC Color reaction with ninhydrin Fluorescamine

40. Determination of amino acid composition

41. Quantification of amino acids

42. fluorescent derivative of amino acid

43. Amino acid sequence can be determined by automated Edman degradation Automated sequencer

44. Edman degradation sequentially removes one residue at a time

46. phenythiocarbamoyl derivative Under a mildly acidic condition Phenylthiohydantoin (PTH)-amino acid

47. Separation of PTH-amino acids

48. Proteins can be specifically cleaved into small peptides to facilitate analysis Divide and conquer CNBr, trysin Overlap peptide Reducing agent: DTT Alkylating agent: iodoacetate Diagonal electrophoresis

49. Cleavage by CNBr

50. Cleavage by Trysin

52. Overlap peptides

53. Disulfide bond reduction

56. Oxidation of cystine

57. Diagonal electrophoresis

58. Amino acid sequences are sources of many kinds of insight Homology - function Evolutionary pathway Internal repeats Localization signal Generation of antibodies Generation of DNA probes

59. Repeating motifs

60. Recombinant DNA technology has revolutionized protein sequencing Still need to work with isolated proteins Genomic and proteomic analyses are complementary

61. DNA sequence yields the amino acid sequence

62. 3.3 Immunology provides important techniques with which to investigate proteins Antigen and antibody Antigenic determinant or epitope Hapten plus macromolecular carrier antiserum Monoclonal and polyclonal antibodies Antibodies to specific proteins can be generated

63. Antibody structure fragment crystallizable

65. Antigen antibody interaction

69. Multiple myeloma generates a large number of cells of a single kind A clone producing immunoglobulin of a single kind Fusion of a short-lived antibody-producing cell with an immortal myeloma cell Hybridoma cells Monoclonal antibodies in clinical assays and affinity purification Monoclonal antibodies with virtually any desired specificity can be readily prepared

71. Staining with a fluorescent-labeled monoclonal antibody

72. enzyme-linked immunosorbent assay (ELISA) Indirect ELISA: detection of antibody Sandwich ELISA: detection and quantitation of antigen Proteins can be detected and quantitated by using an enzyme-linked immunosorbent assay

73. ELISA

76. Western blotting permits the detection of proteins separated by gel electrophoresis

77. Western blotting

78. Fluorescence-labeled antibodies – fluorescence microscopy GFP (green fluorescent protein) fusion Immunoelectron microscopy – gold conjugated antibodies Fluorescent markers make possible the visualization of proteins in the cell

79. Fluorescence microscopy

80. Steroid receptor-GFP fusion protein

82. Immunoelectron microscopy

83. Synthetic peptide: antigen, affinity tag or bait, drug, study of 3D structure Blocking of amino group: t-Boc Activation of carboxyl group: dicyclohexylcarbodiimide (DCC) 3.4 Peptides can be synthesized by automated solid-phase methods Solid-phase method

84. Released in the breakdown of bacterial proteins Useful in identifying the cell surface receptor

85. Synthetic peptides as drugs

87. More stable analog

88. Blocking of amino group: t-Boc Activation of carboxyl group: dicyclohexylcarbodiimide (DCC)

89. Amino acid activation

93. Solid-phase peptide synthesis

97. The mass of a protein can be precisely determined by mass spectrometer Matrix-assisted laser desortion/ionization (MALDI) Electrospray ionization (ESI) Time of flight MALDI-TOF 3.5 Mass spectrometry provides powerful tools for protein characterization and identification

100. Individual components of large complexes can be identified by MALDI-TOF mass spectrometry Peptide mass fingerprinting (PMF) – extensively used in proteomics

102. NMR spectroscopy: structure in solution X-ray crystalogrphy: solid 3.6 Three dimensional protein structure can be determined by NMR spectroscopy and X-ray crystalogrphy

103. Protein crystal, source of X-ray, detector Electron scatters x-rays The scattered waves recombine X-ray crystalography reveals three dimensional structure in atomic detail The atomic arrangement affects the scattering pattern Electron density map

109. Spin state of hydrogen nucleus Resonance Shielding by flow of electrons – chemical shift Nuclear magnetic resonance spectroscopy can reveal The structures of proteins in solution 1D NMR Transfer of magnetization: Nuclear Overhauser effect (NOE), an interaction between nuclei is proportional to the inverse sixth power of the distance Nuclear Overhauser enhancement spectroscopy (NOESY) Off-diagonal peaks identify pairs of protons that are less than 5A apart