1. Biochemistry 412 Analytical & Preparative Protein Chemistry I 1 February 2005

2. Positively-charged basic residues (K, R, & H) Negatively-charged acidic residues (E & D) Hydrophobic “patch” Ligand binding pocket (active site) ca. 40 Å Macromolecular dimensions: Proteins are Amphiphilic Macro-Ions >>> The charged groups, hydrophobic regions, size, and solvation affect the biophysical properties of the protein and largely determine its purification behavior.

3. Amino Acid Side Chains that are Negatively Charged At neutral pH: At pH > 9: Adapted from T. E. Creighton, Proteins W.H.Freeman, 1984

4. Amino Acid Side Chains that are Positively Charged At neutral pH:

5. Water forms a hydration shell around proteins. The properties of this bound water are still the subject of many experimental and theoretical investigations.

6. Makarov et al (1998) Biopolymers 45, 469.

7. Makarov et al (2000) Biophys. J. 76, 2966.

8. Makarov et al (2002) Acc. Chem. Res. 35, 376.

9. Purification schemes vary, depending on the source of the protein and its intrinsic biophysical properties... …some flow-charts for typical schemes follow.

10. Purification Scheme for Proteins from their Natural Source

11. Purification Scheme for Soluble Recombinant Proteins

12. Purification Scheme for Insoluble Recombinant Proteins

13. Purification Scheme for Membrane-Associated Proteins

14. But first some theory…. We need to delve a bit more deeply into the hydrodynamic properties of proteins so that you understand why things work the way they do

15. Adapted from T. E. Creighton, Proteins, W.H.Freeman,1984.

16. Adapted from T. E. Creighton, Proteins W.H.Freeman, 1984

17. Adapted from T. E. Creighton, Proteins, W.H.Freeman,1984. 1/2 is the root-mean-square (rms) average end-to-end distance of the polypeptide chain. R G, the radius of gyration, is the rms distance of the collection of atoms from their common center of gravity. 2 ≈ /6 for large polymers.

18. Adapted from T. E. Creighton, Proteins, W.H.Freeman,1984.

19. Translational Diffusion of Macromolecules Adapted from T. E. Creighton, Proteins, W.H.Freeman,1984. (5-20) Q: can anyone guess why people are celebrating about this this year?

20. Some Examples of Diffusion Coefficients Adapted from T. E. Creighton, Proteins, W.H.Freeman,1984.

21. Therefore, an average, garden- variety protein with a diffusion coefficient of 10 -6 cm 2 /sec, will diffuse approximately 10 5 Å in 1 sec. 10 5 Å (= 10 -5 m = 10  m) is approximately the diameter of an average human cell.

22. Adapted from T. E. Creighton, Proteins, W.H.Freeman,1984.

23. Length Dependence of the Radius of Gyration of Polypeptides Adapted from T. E. Creighton, Proteins W.H.Freeman, 1984

24. Adapted from T. E. Creighton, Proteins, W.H.Freeman,1984.

25. Enough with the theory!! How do I purify a protein?