1. Fundamentals of Biochemistry
Third Edition
Donald Voet • Judith G. Voet • Charlotte W. Pratt
Chapter 5
Proteins: Primary Structure
Copyright © 2008 by John Wiley & Sons, Inc.

2. Section 1 – Polypeptide Diversity
Insulin primary structure
Primary Structure – linear sequence of amino acids in a polypeptide chain
20100 = 1.27 x possible combinations
9 x 1078 atoms estimated in universe

4. Section 2 – Protein Purification
Purification was difficult for a endogenous protein
First proteins studies were very abundant
Modern cloning techniques all for production of large quantities of specific proteins
This process still requires that the protein be isolated from a cell, and purified from the other cellular components

5. Conditions affect protein StabilitypH
The wrong pH causes denaturation
Temperature
The wrong temperature can cause denaturation
Presence of other proteins
Proteases can destroy proteins
Adsorption to surfaces
Some proteins can be denatured upon exposure to air
Long term storage
Most proteins should be stored at -20°C or lower to minimize degradation and denaturation

6. ELISA
Enzyme linked immunosorbent assay
Used to determine (quantify) the amount of protein present
Animation

7. Spectroscopic method for determining protein concentration
Beer-Lambert law
A=εcl
A280 – absorbance of F, Y, W

8. Colorimetric method for determining protein concentration
Bradford assay

10. Salting Out

11. Ion Exchange Chromatography
Animation

12. Gel Filtration Chromatography
Animation

13. Affinity Chromatography
Immunoaffinity
Metal chelate

14. SDS-PAGE
Sodium-dodecyl sulfate – Poly acrylamide gel electrophoresis

15. Capillary Electrophoresis 2D Gel electrophoresis

16. Section 3 – Protein Sequencing
Important to know the sequence of a protein
Primary structure dictates shape
Evolutionary comparisons can be made
Diseases arise from mutations of primary structure

18. Step 1.

19. Step 2

20. Step 3

22. Step 4
Edman
Degradation
Animation

25. Section 4 – Protein Evolution

26. Protein Evolution
Homologue – evolutionarily similar proteins within the same species
Invariant residue – identical aa among homologues
Conserved residue – similar (class) aa among homologues
Hypervariable residue – no similarity among homologues

27. Protein Evolution
Domain – region of proteins that have very similar folding patterns ( aa)
Orthologues – homologous proteins in different species
Paralogues – independently evolving genes in the same species
Pseudogenes – duplicated gene that are not expressed

29. All problems at end of chapter except 6, 13, and 19

30. You have isolated a decapeptide (10 residues) called FP, which has anticancer activity. Determine the sequence of the peptide from the following information.
1. One cycle fo Edman degradation of intact FP yields 2 mole of PTH-aspartate per mole of FP.
Treatment of a solution of FP with 2-ME followed by the addition of trypsin yields three peptides
(Ala, Cys, Phe) (Arg, Asp) (Asp, Cys, Gly, Met, Phe)
The intact (Ala, Cys, Phe) peptide yields PTH-cysteine in the first cycle of Edman degradation.
3. Treatment of 1 mol of FP with carboxypeptidase (cleaves at C-terminus of all residues) yields 2 mol of phenylalanine.
4. Treatment of intact pentapeptide (Asp, Cys, Gly, Met, Phe) with BrCN yields two peptides with composition (homoserine lactone, Asp) and (Cys, Gly, Phe) The (Cys, Gly, Phe) peptide yields PTH-glycine in the first cycle of Edman degration.