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Exploring Proteins and Proteomes. Stryer,CHAPTER 3 ppt

  1. BS(integrated) BIOCHEMISTRY Email ID:khair_awkum@yahoo.com Biochemistry Sixth Edition By Khair Ullah Chapter 3: Exploring Proteins and Proteomes
  2. Methods in Protein Chemistry These are methods used in isolation, purification, detection, degradation, analysis and synthesis of proteins. As one would expect, most of these involve aqueous media and require a knowledge of pH, pKas, and charge on a peptide at various pH values. Proteome: defines the compete functional information about a group of proteins that work together as a functional unit.
  3. Assay of an Enzyme In this reaction, the increase in absorbance of NADH at 340 nm is used to follow the formation of pyruvate This is an oxidation-reduction reaction
  4. Enzyme Activity Total activity in soution use Unit of enzyme activity: µmol substrate/min or mol S/sec = katal To follow during purification use Specific activity: µmol substrate/min-mg E or mol S/sec-kg E = katal/kg E To compare different enzymes use Molecular activity also called turn-over number (TON): µmol substrate/min-µmol E or mol S/sec-mol E = katal/mol E
  5. Centrifugation Separation of a cell homogenate.
  6. Solubility of Proteins Salting in: When proteins are placed in an aqueous solution, the only ionic species in solution are the other protein molecules. Water, although polar, is only slightly ionized so the proteins tend to aggregate based on ionic interactions that form between themselves. The interactions between protein molecules are more favorable than interactions between water and a protein. At low salt concentration (NaCl), other ionic species are now present to compete with the ionic protein:protein interactions. As a result, the ionic interactions between proteins break up and the proteins dissolve. Both the small ions (from NaCl)
  7. Solubility of Proteins Salting out: At high salt concentration (typically with (NH4)2SO4 or Na2SO4), water molecules are more strongly attracted to these small ions (especially multivalent ions) than to the large protein molecules. The proteins are left then to seek whatever favorable interactions exist and these are the protein:protein associations which result in aggregation and precipitation. Isoelectric precipitation: At the pI there is zero net charge on a protein. At a pH away from the pI, each protein molecule bears an identical charge (either + or - depending on the pH) resulting in repulsion between molecules. At the pI, no repulsion occurs, and the proteins will aggregate and precipitate.
  8. Determining the isoelectric point (pI) Isoelectric point: The pI is the pH at which there is zero net charge on a molecule. Look at Asp. The zero net charge form is a part of the first two ionizations. Therefore, the maximum amount of this is present at a pH of (2.09 + 3.86)/2 = 2.98 = pI. HOOC-CH2-CH-COOH NH3 + HOOC-CH2-CH-COO - NH3 + + H+ 2.09 HOOC-CH2-CH-COO - NH3 + - OOC-CH2-CH-COO - NH3 + - OOC-CH2-CH-COO - NH3 + - OOC-CH2-CH-COO - NH2 + H+ 3.86 + H+ 9.82
  9. Dialysis Separation of very large from very small molecules is based on an attempt to equilibrate concentration. Osmotic pressure
  10. Gel Filtration – size separation
  11. Affinity Chromatography
  12. HPLC (up to 5000 psi) Proteins can be detected from absorbance of the peptide bonds in the uv at 220 nm. However, it is more commonly done at 280 nm as seen earlier.
  13. Ion Exchange Chromatography Depends upon the charge on each of the various molecules being separated.
  14. Ion Exchange Resins
  15. Determining the Charge on a Peptide Determing the charge on a peptide involves a knowledge of ionic equilibria, pKas and the ionic forms present at a given pH. In a peptide, the amino terminus, the carboxy terminus and the ionizable side chains may be charged at a given pH. The sum of these charges gives the net peptide charge.
  16. Electrophoresis Separation of molecules by electrophoresis depends upon: 1. the strength of the electric field (voltage), 2. the charge on each molecule, 3. the frictional coefficient of movement through the solid support which in turn depends on the radius or mass of the molecule. So, essentially electrophoresis separates based on a charge/mass ratio.
  17. Electrophoresis A classical electrophoresis apparatus. V buffer buffer solid support for sample cooling plate
  18. Sodium Dodecyl Sulfate SDS is an anionic detergent that binds uniformly along a protein chain. About one SDS binds for every two amino acid residues. Thus all proteins bear the same charge/mass ratio and separation by electrophoresis will be based on mass alone.
  19. SDS Gel Electrophoresis
  20. Proteins after Staining
  21. Isoelectric Focusing Electrophoresis on a mixture of polyampholytes. Each molecule migrates to its point of zero net charge.
  22. SDS PAGE after IEF
  23. Sequential Purification Steps
  24. SDS electrophoresis
  25. Ultracentrifugation The S value is a measure of the rate of sedimentation, (a sedimentation coefficient) and is not linear with MW because of molecular shape.
  26. In a sucrose or cesium chloride gradient a molecule migrates the buoyant density equal to its own.
  27. Amino Acid Analysis Determines amino acid composition of a protein. A protein is hydrolyzed in 6N HCl, 24 hrs at 100o C. Separation of AA by ion exchange chromatography.
  28. Detecting Amino Acids Classical reagent for amino acids. Reaction requires 2-5 min at 100o C and gives nanomole level detection. Ruhemann’s Purple 570 nm OH O O OH NH2-CH-COOH CH3 + O O O O NCO2 CH3 CHO + +2
  29. Detecting Amino Acids Reacts immediately with primary amines at room temperature. Detection at picomol level due to fluorescent products
  30. Automated Sequencing
  31. Edman’s Method Edman degradation procedure - Determining one residue at a time from the N-terminus (1) Treat peptide with phenyisothiocyanate (PITC) at pH 9.0 which reacts with the N-terminus to form a phenythiocarbonyl (PTC)-peptide. (2) Treat the PTC-peptide with anh. trifluoroacetic acid (TFA) to selectively cleave the N-terminal peptide bond and form a triazolinone derivative. (3) Extract N-terminal derivative from the peptide. (4) Rearrange to a phenylthiohydantoin (PTH)-amino acid with aq. HCl then chromatograph.
  32. Edman’s Method Phenylthio- hydantoin (PTH) PITC
  33. Edman’s Method N=C=S phenylthiocarbamyl-peptide + NH2 - CH - C - N - CH -C O H O CH2-OH CH3 NH - C - S NH - CH - C - N - CH -C O H O CH2-OH CH3 S N O CH3NH - + N N O CH3 S NH2 - CH - C O CH2-OH thiazolinone derivativephenylthiohydantoin derivative
  34. Separation of PTH-AAs by HPLC
  35. N-Terminal Reagents DNFB - Sanger’s reagent (dinitrofluorobenzene) DANSYL choride (dimethylaminonaphthalenesulfonyl chloride) NO2 NO2 F DNFB SO2Cl N(CH3)2 DANSYL-Cl
  36. Other Reagents C-terminal: Hydrazine Disulfide reduction: Dithiothreitol - Cleland’s Reagent Thiols: Iodoacetate 5,5’-dithiobis-(2-nitrobenzoic acid) - Ellman’s reagent NH2-NH2 HO C H H C OH CH2 -SH CH2 -SH Cleland's NO2 S S COOH NO2 COOHEllman's I-CH2-COOH
  37. Protein Cleavage Protein sequencing is most manageable with small polypeptides. Therefore, in order to sequence a large protein, it must be cleaved into smaller pieces. Cleavage is conducted using either chemical or enzymatic methods. The pieces must be separated and purified before sequencing.
  38. Chemical and Enzymatic Cleavage
  39. CNBr Cleavage at Met
  40. CNBr Cleavage at Met
  41. Enzymatic cleavage by Trypsin
  42. An Example, Peptide overlap
  43. Dithiothreitol Reduction of –S-S-
  44. Iodoacetate reaction with -SH
  45. Performic acid oxidation of –S-S-
  46. Sequencing using DNA Edman’s degradation has been a tremendous asset in protein sequencing, however, for larger proteins recombinant DNA technology is now being used.
  47. Merrifield Solid-Phase Synthesis Merrifield and coworkers at Rockefeller Institute devised a revolutionary solid phase method of protein synthesis in 1963. The proved that this method was effective for larger proteins by synthesizing ribonuclease, an enzyme with 124 amino acid residues. A chloromethylated polystyrene polymer (resin) was used as the solid support. Dicyclohexylcarbodiimide was used as an amino acid activator and a water scavenger in the condensation reaction. Merrifield received a Nobel Prize in 1984 for this work.
  48. Other Methodologies Immunochemistry: (omit as this is in section 3.3) ELISA = Enzyme-linked immunosorbent assay Western blotting Fluorescense microscopy Mass Spectrometry: (section 3.5) MALDI = matrix-assisted laser desorption- ionization TOF = time of flight X-ray Crystallography: (section 3.6) Nuclear Magnetic Resonance: (section 3.6) NOESY = nuclear Overhauser enhancement spectroscopy.
  49. • Amino acid sequence data provide a basis for preparing antibodies specific for a protein of interest. • Amino acid sequence are valuable for making DNA probes that are specific for the genes encoding the corresponding proteins. • The nucleotide sequence of DNA (gene) directly reveals the entire amino acid sequence of the protein encoded by the gene. • However, DNA sequence can not disclose the information regarding post- translational modification. Practical Usage of Amino Acid and DNA Sequences
  50. Antibody • Antibody (immunoglobulin) is a protein synthesized by an animal in response to the presence of a foreign substance (antigen). • Antibodies have specific and high affinity against antigens. • Proteins, polysaccharides and nucleic acids can be effective antigens. • Epitope : a specific group or cluster (portion) of antigen to stimulate the synthesis of an antibody and recognized by a specific antibody (antigenic determinant) • Hapten : a small molecule containing epitope attached to a carrier
  51. Antibody (continued) • Each antibody producing cell synthesizes only one type of antibody recognizing a single kind of epitope. • The proliferation of a given antibody producing cell is stimulated by the binding of its designated antigen to the cell surface receptor of the antibody producing cell . • Periodic injections of an antigen into the host animal can raise the antibodies specifically recognizing the injected foreign substance. • Blood withdrawn from the immunized host animal  centrifugation  separation of blood cells (pellet) and serum (supernatant)  anti-serum • Anti-serum contains multiple kinds of antibodies each recognizing a different surface feature of the same antigen. • This heterogenic antibodies are called as polyclonal
  52. Monoclonal Antibody • Monoclonal hybridoma cell lines can generate large amount of homogeneous antibodies. • Monoclonal antibodies can serve as precise analytical, preparative and therapeutic reagents.(HCV, HIV, herceptin) Immuno- Staining of Drosophila Embryo using Monoclonal Antibody against Engrailed Plasma cell by antigen-antibody interaction
  53. Monoclonal antibody drugs?
  54. Herceptin Binds to the C-terminus of Domain Herceptin Fab I III II IV N C HER2
  55. Ribbon Diagram of Her-3 ECD N C “Tethered” I II III IV Right-handed β helix Laminin-like folds Fig. 14.28 pp397
  56. Surface representations of EGFR and HER2 in Antibody-Bound Conformations Herceptin
  57. ELISA (Enzyme-Linked Immuno-Sorbent Assay) Antibody detection, anti-HIV antibody Antigen detection
  58. Western Blotting Radioactive secondary antibody For protein expression and purification
  59. Immuno-Fluorescence Microscopy Actin Filament Staining using α-actin antibody Immuno-Electron Microscopy Detection of a channel protein from the synaptic vesicles using antibodies tagged with electron-dense markers such as gold or ferritin (Resolution better than 10 nm) Fluorescence-labeled antibodies (resolution 200nm) ex) Glucocorticoid receptor
  60. Time of Flight Mass Spectrometer
  61. Nuclear Magnetic Resonance
  62. End of Chapter 3 Thank You

Notas do Editor

  1. Myeloma cell : 골수세포 Spleen cell : 지라(비장), 콩팥 위에 위치 Pancreas : 이자 (췌장)
  2. Herceptin 2011 sale: $1.7 billion
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