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Biochemistry 412 2004 February 17th Lecture Analytical & Preparative Protein Chemistry I

Biochemistry 412 2004 February 17th Lecture Analytical & Preparative Protein Chemistry I. Proteins are Amphiphilic Macro-Ions. Positively-charged basic residues (K, R, & H). Hydrophobic “patch”. Macromolecular dimensions:. ca. 40 Å. Ligand binding pocket (active site).

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Biochemistry 412 2004 February 17th Lecture Analytical & Preparative Protein Chemistry I

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  1. Biochemistry 412 2004 February 17th Lecture Analytical & Preparative Protein Chemistry I

  2. Proteins are Amphiphilic Macro-Ions Positively-charged basic residues (K, R, & H) Hydrophobic “patch” Macromolecular dimensions: ca. 40 Å Ligand binding pocket (active site) Negatively-charged acidic residues (E & D) >>> 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) Biopolymers45, 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. <r2>1/2 is the root-mean-square (rms) average end-to-end distance of the polypeptide chain. RG, the radius of gyration, is the rms distance of the collection of atoms from their common center of gravity. <RG>2 ≈ <r2>/6 for large polymers. Adapted from T. E. Creighton, Proteins, W.H.Freeman,1984.

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

  19. Translational Diffusion of Macromolecules (5-20) Adapted from T. E. Creighton, Proteins, W.H.Freeman,1984.

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

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

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

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

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

  25. Chromatography Sample containing proteins or peptides Liquid flow Liquid flow Separation according to: -molecular weight/ size -charge -hydrophobicity -affinity Time 1 2 3 4 5 4:37 990909

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