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Amino Acids and Proteins

Amino Acids and Proteins. 3-D Structure of Myoglobin. 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)

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Amino Acids and Proteins

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  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)

  5. Structure of -amino acids

  6. The 20 Amino Acids Found in Proteins

  7. Properties of Different Amino Acid Side Chains

  8. Stereochemistry of -amino acids

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

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

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

  12. 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 (g-Glu-Cys-Gly) makes the inside of the cell a highly reducing environment.

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

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

  15. Modified or Unusual Amino Acids

  16. Absorption of UV Light by Aromatic Amino Acids

  17. 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

  18. Formation of a Peptide

  19. Planarity of Peptide (Amide) Bond

  20. .

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

  22. Examples of Oligopeptides

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

  24. 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).

  25. Evolution and Conservation of Protein Sequences Translation elongation factor Tu/1a Myoglobin

  26. The Genetic Code

  27. DNA RNA Protein

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

  29. Charging of tRNAs with Specific Amino Acids

  30. Translation of mRNA into Protein

  31. 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.

  32. Disulfide Bond Formation in Insulin

  33. Methods in Protein Biochemistry

  34. Gel Electrophoresis

  35. Polyampholyte Character of a Tetrapeptide and Isoelectric Points Group pKa a-NH3+ 9.7 Glu g-COOH 4.2 Lys e-NH3+ 10.0 a-COOH 2.2 Isoelectric Point (pI), pH at which molecule has net zero charge, determined using computer program for known sequence or empirically (by isoelectric focusing).

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

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

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

  39. Centrifugation Low-speed, high-speed, or ultracentrifugation: different spin speeds and g forces • 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

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