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. Primary structure of proteins = amino acid sequence
By convention, always written N (amine) terminus to C (carboxylic acid) terminus
Bovine insulin: 1 protein, 2 chains (subunits) held together by two disulfide bridges between cysteine side chains (Sanger and Tuppy, Biochem. J. 49 (4): 463–81, 1951)

3. Table 5-1

4. Proteins function when their primary, secondary, tertiary and (if applicable) quaternary structures are intact. If any of these are lost, the protein is denatured. Denaturation is reversible, in most cases.
Protein stability is dependent on:

5. Proteins function when their primary, secondary, tertiary and (if applicable) quaternary structures are intact. If any of these are lost, the protein is denatured. Denaturation is reversible, in most cases.
Protein stability is dependent on:
• pH
• temperature
• concentrations of ions in solution
• absence of proteases and surfaces

6. Quantifying protein concentration
ELISA: Enzyme-linked immunosorbent assay

7. Quantifying protein concentration
Proteins absorb in the ultraviolet part of the EM spectrum. So, in principle, a spectrometer may be used to quantify protein concentration at 280 nm (aromatic rings) or 200 nm (peptide bond). Problem: nucleic acids absorb at 260 nm.

8. Purifying proteins

9. Purifying proteins: “salting out”

10. Purifying proteins: chromatography and the isoelectric point

11. Purifying proteins: ion exchange chromatography
By changing the pH or ionic strength of the eluting solution, a mixture of proteins may be separated

12. Purifying proteins: gel filtration chromatography
This separates by molecular size

13. Purifying proteins: affinity chromatography
For instance, I used lentil lectin-infused beads as the stationary phase to purify a glycoprotein in a protein mixture, because of the lectin’s affinity for certain sugars.

14. Purifying proteins: SDS-PAGE electrophoresis
SDS = sodium dodecyl sulfate (a surfactant that denatures proteins into long strands)
PAGE = polyacrylamide (the matrix material) gel electrophoresis

15. Purifying proteins: electrophoresis

16. Purifying proteins: electrophoresis AND isoelectric focusing: 2D gel electrophoresis

17. Purifying proteins: ultracentrifugation
By spinning a protein mixture in an ultracentrifuge (>100,000 g), you can get proteins to settle at the bottom of a centrifuge tube. The rate at which it does so is quantified by the sedimentation coefficient, which is measured in Svedbergs (S), which is a unit equal to 10–13 seconds.

18. Purifying proteins: ultracentrifugation
By spinning a protein mixture in an ultracentrifuge (>100,000 g), you can get proteins to settle at the bottom of a centrifuge tube. The rate at which it does so is quantified by the sedimentation coefficient, which is measured in Svedbergs (S), which is a unit equal to 10–13 seconds.
Yes, this is the same “S” found in, say, organelle subunits, like the 16S subunit of the bacterial ribosome.

19. Protein sequencing

20. Protein sequencing: Sanger method
Upon hydrolysis, the N-terminal amino acid has a chromophore attached, which can be separated and identified
Box 5-1

21. Protein sequencing: Dansyl chloride
Similar to the Sanger method – N-terminus residue

22. Protein sequencing:
Cleave the disulfide bridge to free sub-units

23. Protein sequencing: Prevent the disulfide bridge from reforming by preventing oxidation of thiol

24. Protein sequencing: Selective cleaving of peptide bonds
Trypsin is an enzyme that cleaves the peptide bond at the C-terminus of lysine or arginine (if the N-terminus is not a proline)

25. endo- = within; exo- = outside of

26. Protein sequencing: Edman degradation
Can remove up to 100 residues from the N-terminus, one at a time
Protein sequencing: Edman degradation
Pehr Edman (1950)

27. Protein sequencing: Mass spectrometry
After selective cleavage of proteins, electrospray ionization mass spectrometry allows for the rapid calculation of fragment size

28. Protein sequencing: Mass spectrometry sample output
Sample calculation 5-1 shows the original protein to have a mass of 16,975 D

29. Protein sequencing:
Tandem mass spectrometry

30. Protein sequencing:
Aligning the fragments

31. Protein sequencing: The placement of disulfide bridges can be determined

32. Proteomics
Also, the BioSample database at ncbi.nlm.nih.gov

33. Figure 5-20

34. Table 5-5 part 1

35. Table 5-5 part 2

36. Cytochrome c in different organisms: numbers indicate differences per 100 residues

37. Human hemoglobin = α2 β2

38. What does a steep slope mean?

39. Proteins evolve by repeating or adding domains