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Amino Acids and the Primary Structure of Proteins

Amino Acids and the Primary Structure of Proteins. Important biological functions of proteins 1. Enzymes, the biochemical catalysts 2. Storage and transport of biochemical molecules 3. Physical cell support and shape (tubulin, actin, collagen)

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Amino Acids and the Primary Structure of Proteins

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  1. Amino Acids and the Primary Structure of Proteins Important biological functions of proteins 1. Enzymes, the biochemical catalysts 2. Storage and transport of biochemical molecules 3. Physical cell support and shape (tubulin, actin, collagen) 4. Mechanical movement (flagella, mitosis, muscles) (continued)

  2. Amino Acids and the Primary Structure of Proteins 5. Decoding cell information (translation, regulation of gene expression) 6. Hormones or hormone receptors (regulation of cellular processes) 7. Other specialized functions (antibodies, toxins etc)

  3. Zwitterionic form of amino acids • Under normal cellular conditions amino acids are zwitterions(dipolar ions): • Amino group = -NH3+ • Carboxyl group = -COO-

  4. Two representations of an amino acid at neutral pH

  5. Titration Curve for Alanine • Titration curves are used to determine pKa values • pK1 = 2.4 • pK2 = 9.9 • pIAla = isoelectric point

  6. Aliphatic R Groups • Glycine (Gly, G) - the a-carbon is not chiral since there are two H’s attached (R=H) • Four amino acids have saturatedsidechains: • Alanine (Ala, A) Valine (Val, V) • Leucine (Leu, L) Isoleucine (Ile, I) • Proline (Pro, P) 3-carbon chain connects a-C and N

  7. Stereoisomers of Isoleucine • Ile has 2 chiral carbons, 4 possible stereoisomers

  8. Aromatic Amino Acids

  9. Methionine and Cysteine

  10. Formation of Cystine

  11. Histidine, Lysine, and Arginine

  12. Aspartate, GlutamateAsparagine, Glutamine

  13. Peptide Chain Nomenclature • Amino acid“residues” compose peptide chains • Peptide chains are numbered from the N (amino) terminus to the C (carboxyl) terminus • Example: (N) Gly-Arg-Phe-Ala-Lys (C) (or GRFAK) • Formation of peptide bonds eliminates the ionizable a-carboxyl and a-amino groups of the free amino acids

  14. Peptide SequencingEdmann Degradation

  15. Edmann Degradation (cont.)

  16. Edmann Degradation (cont.)

  17. Edmann Degradation (cont.)

  18. Cleaving Disulfide bonds andProtecting the thiols formed • Disulfide bonds in proteins must be cleaved: • (1) To permit isolation of the PTH-cysteine during the Edman procedure (2) To separate peptide chains • Treatment with thiol compounds reduces the (R-S-S-R) cystine bond to two cysteine (R-SH) residues • Thiols are protected with iodoacetate

  19. Further Protein Sequencing Strategies • Proteins may be too large to be sequenced completely by the Edman method • Proteases (enzymes cleaving peptide bonds) and chemicalagents are used to selectivelycleave the protein into smaller fragments • Cyanogen bromide (BrCN) cleaves polypeptides at the C-terminus of Met residues

  20. Protease Enzymescleave specific peptide bonds • Chymotrypsin- carbonyl side of aromatic or bulky noncharged aliphatic residues (e.g. Phe, Tyr, Trp, Leu) • Trypsin - carbonyl side, basic residues (Lys,Arg). • Staphylococcus aureus V8 protease - carbonyl side of negatively charged residues (Glu, Asp). NOTE: in 50mM ammonium bicarbonate cleaves only at Glu.

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