1. SURVEY OF BIOCHEMISTRY Amino Acids and Proteins
Begin lecture with a 5-min study exercise on naming amino acids! Student with the highest number correct will get a prize.

2. Amino Acids
Can you give the 1-letter and 3-letter names for all 20 amino acids within 5 minutes?
Can you draw a oligopeptide of any given sequence?
With correct stereochemistry?

3. Amino Acids: Nonpolar Side Chains

4. Amino Acids: Polar, Uncharged Side Chains

5. Cysteine & Disulfide Bonds

6. Cysteine & Disulfide Bonds

7. Amino Acids: Polar, Uncharged Side Chains

8. Amino Acids: Polar, Uncharged Side Chains

9. Amino Acids: Polar, Charged
Be able to draw, name and give 1-letter/3-letter codes for all 20 amino acids!

10. pKa and pI
pI = Isoelectric point the pH at which a molecule carries no net electric charge
pKa2
pKa1
pI = [pKa1 + pKa2]

11. Polypeptide Sequence Nomenclature
Be able to draw a polypeptide!
Tetrapeptide: AYDG

12. Peptide Bond
Peptide bond links one amino acid to another.

13. Torsion Angles in Proteins
Peptides typically assume the trans conformation

14. Torsion Angles Defined…
Psi: angle made from atoms N-Calpha-Ccarbonyl-Nn+1

15. Torsion Angles Defined…
Phi: angle made from atoms Ccarbonyl,n-1-Nn-Calpha-Ccarbonyl,n

16. Hydrogen bonding stabilizes the alpha helical structure!
Alpha Helix
Sequence coils in a right-handed manner. Notice hydrogen bonding along the helical axis from carbonyl oxygen (of residue n) to the amino hydrogen of residue (n+4).
Hydrogen bonding stabilizes the alpha helical structure!

17. Beta Sheets
Notice the hydrogen bonding from the carbonyl oxygen to the amino hydrogen

18. Protein Purification Overview
Courtesy of

19. Salting Out: (NH4)2SO4

20. Gel Filtration Chromatography
Separation based on Size

21. Ion Exchange Chromatography
Separation based on Charge

22. Affinity Chromatography
What could be to cause proteins to elute off of an affinity column?

23. Ways to Assess Protein Purification
Total Protein Concentration Assays
Beer’s Law
Colorimetric Assays
Specific Protein Assays
Activity Assays
Immunoassays
ELISA
RIA

24. Beer’s Law
What is the molar concentration of a solution of Bovine Serum Albumin (BSA) that exhibits an A280 of 0.75 with a path length of 1 cm?

25. Beer’s Law
What is the molar concentration of a solution of Bovine Serum Albumin (BSA) that exhibits an A280 of 0.75 with a path length of 1 cm?
Must know the value of
0.75
Conc. =
(43,824 M-1cm-1) x (1 cm)
Conc. = M = 17 µM

26. Bradford Protein Assay
Suppose you have a protein mixture and you need to determine the protein concentration.
You cannot use Beer’s Law. Because you would not know the extinction coefficient for the protein mixture at 280 nm
Alternative approach: Bradford Protein Assay

27. Bradford Protein Assays
Bradford Protein Assay reagent contains Coomassie brilliant blue which reacts with basic (esp. Arg) and aromatic amino acids to yield a blue color with intensity proportional to the protein concentration.
ENDED HERE…
How is data generated?
How is data analyzed?

28. Data Generation: Bradford Assay
Reference contains only buffer
Create a series of protein standards at known increasing concentrations and mix with the Bradford reagent
µg/mL
Treat the protein mixture of unknown concentration in the same manner as the standards and compare - visually and spectrophotometrically!

29. Bradford Assay Data Analysis
Graph the absorbance at 595 nm as a function of [standard] in µg/mL.
Fit the line and use it to deduce the [protein] in the mixture based on its absorbance.