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 saturated side chains:
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, Glutamate Asparagine, 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 Sequencing Edmann Degradation

15. Edmann Degradation (cont.)

16. Edmann Degradation (cont.)

17. Edmann Degradation (cont.)

18. Cleaving Disulfide bonds and Protecting 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 chemical agents are used to selectively cleave the protein into smaller fragments
Cyanogen bromide (BrCN) cleaves polypeptides at the C-terminus of Met residues

20. Protease Enzymes cleave 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.