1. Exam I Review I

2. Several Amino Acids Occur Rarely in Proteins Figure 4.4 (c) Several amino acids that act as neurotransmitters and hormones.

3. pK a Values of the Amino Acids You should know these numbers and know what they mean Arginine, Arg, R: pK a (guanidino group) = 12.5 Aspartic Acid, Asp, D: pK a = 3.9 Cysteine, Cys, C: pK a = 8.3 Glutamic Acid, Glu, E: pK a = 4.3 Histidine, His, H: pK a = 6.0

4. pK a Values of the Amino Acids You should know these numbers and know what they mean Lysine, Lys, K: pK a = 10.5 Serine, Ser, S: pK a = 13 Threonine, Thr, T: pK a = 13 Tyrosine, Tyr, Y: pK a = 10.1

5. A Sample Calculation What is the pH of a glutamic acid solution if the alpha carboxyl is 1/4 dissociated? pH = 2 + (-0.477) pH = 1.523 Note that, when the group is ¼ dissociated, 1/4 is dissociated and ¾ are not; thus the ratio in the log term is ¼ over ¾ or 1/3.

6. Another Sample Calculation What is the pH of a lysine solution if the side chain amino group is 3/4 dissociated? pH = 10.5 + (0.477) pH = 10.977 = 11.0 Note that, when the group is ¾ dissociated, ¾ is dissociated and ¼ is not; thus the ratio in the log term is ¾ over ¼ or 3/1.

7. 5.1 What Architectural Arrangements Characterize Protein Structure? Proteins are classed according to shape and and solubility Shape - globular or fibrous The four levels of protein structure are: - Primary - sequence - Secondary - local structures - H-bonds - Tertiary - overall 3-dimensional shape - Quaternary - subunit organization

8. Estimation of Protein Concentrations Several methods rely on reduction of Cu 2+ to Cu + to estimate protein. BCA forms a purple complex with Cu +.

10. Enzymatic Fragmentation Trypsin - cleavage on the C-side of Lys, Arg Chymotrypsin - C-side of Phe, Tyr, Trp Clostripain - like trypsin, but attacks Arg more than Lys Staphylococcal protease – C-side of Glu, Asp in phosphate buffer – specific for Glu in acetate or bicarbonate buffer

11. Chemical Fragmentation Cyanogen bromide CNBr acts only on methionine residues CNBr is useful because proteins usually have only a few Met residues See Fig. 5.11 for mechanism Be able to recognize the results – a peptide with a C-terminal homoserine lactone

12. Reconstructing a Sequence Compare cleavage by trypsin and staphylococcal protease on an unknown peptide: Trypsin cleavage of the unknown peptide gave: A-E-F-S-G-I-T-P-K L-V-G-K Staphylococcal protease cleavage gave: F-S-G-I-T-P-K L-V-G-K-A-E

13. Reconstructing the Sequence of an Unknown Peptide Overlap of the two sets of fragments: L-V-G-K A-E-F-S-G-I-T-P-K L-V-G-K-A-E F-S-G-I-T-P-K Correct sequence: L-V-G-K-A-E-F-S-G-I-T-P-K

14. 5.7 Do Proteins Have Chemical Groups Other Than Amino Acids?

15. Ion Exchange Chromatography Can Be Used to Separate Molecules on the Basis of Charge Figure 5A.3 The separation of amino acids on a cation exchange column.

16. Gel Filtration Chromatography Can Be Used to Separate Molecules on the Basis of Size Figure 5A.4 (a) A gel filtration chromatography column. Larger molecules are excluded from the gel beads and emerge from the column sooner than smaller molecules, whose migration is retarded because they can enter the beads. (b) An elution profile.

17. SDS-Polyacrylamide Gel Electrophoresis (SDS- PAGE) Figure 5A.5 The structure of sodium dodecyl sulfate (SDS).

18. Affinity Chromatography Figure 5A.8 Diagram illustrating affinity chromatography. Small molecule targets (ligands) are immobilized through covalent attachment to an insoluble matrix, such as cellulose or polyacrylamide, in a column. The protein of interest is then passed through the column, where it binds to the target ligands, while other proteins pass through without binding. The protein can be eluted by addition of high concentrations of the free ligand.

19. 6.1 What Noncovalent Interactions Stabilize the Higher Levels of Protein Structures? What are these “weak forces”? What are the relevant numbers? van der Waals: 0.4 - 4 kJ/mol hydrogen bonds: 12-30 kJ/mol ionic bonds: 20 kJ/mol hydrophobic interactions: <40 kJ/mol

20. 6.1 What Noncovalent Interactions Stabilize the Higher Levels of Protein Structure? Secondary, tertiary, and quaternary structure of proteins is formed and stabilized by weak forces Hydrogen bonds are formed wherever possible Hydrophobic interactions drive protein folding Ionic interactions usually occur on the protein surface Van der Waals interactions are ubiquitous

21. Exposed N-H and C=O groups at the ends of an α-Helix can be “capped”. Figure 6.9 Four N-H groups at the N- terminal end of an α-helix and four C=O groups at the C-terminal end lack partners for H-bond formation. The formation of H bonds with other nearby donor and acceptor groups is referred to as helix capping. Capping may also involve appropriate hydrophobic interactions that accommodate nonpolar side chains at the ends of helical segments.

22. The β-Pleated Sheet Figure 6.10 A “pleated sheet” of paper with an antiparallel β- sheet drawn on it.

23. The β-Turn Figure 6.12 The structures of two kinds of β-turns (also called tight turns or β-bends). Four residues are required to form a β-turn. Left: Type I; right: Type II.