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Biochemistry 412 Analytical & Preparative Protein Chemistry I 1 February 2005 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. Amino Acid Side Chains that are Negatively Charged At neutral pH: At pH > 9: Adapted from T. E. Creighton, Proteins W.H.Freeman, 1984 Amino Acid Side Chains that are Positively Charged At neutral pH: Water forms a hydration shell around proteins. The properties of this bound water are still the subject of many experimental and theoretical investigations. Makarov et al (1998) Biopolymers 45, 469. Makarov et al (2000) Biophys. J. 76, 2966. Makarov et al (2002) Acc. Chem. Res. 35, 376. Purification schemes vary, depending on the source of the protein and its intrinsic biophysical properties... …some flow-charts for typical schemes follow. Purification Scheme for Proteins from their Natural Source Purification Scheme for Soluble Recombinant Proteins Purification Scheme for Insoluble Recombinant Proteins Purification Scheme for Membrane-Associated Proteins 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 Adapted from T. E. Creighton, Proteins, W.H.Freeman,1984. Adapted from T. E. Creighton, Proteins W.H.Freeman, 1984 <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. Adapted from T. E. Creighton, Proteins, W.H.Freeman,1984. Translational Diffusion of Macromolecules (5-20) Q: can anyone guess why people are celebrating about this this year? Adapted from T. E. Creighton, Proteins, W.H.Freeman,1984. Some Examples of Diffusion Coefficients Adapted from T. E. Creighton, Proteins, W.H.Freeman,1984. Therefore, an average, gardenvariety protein with a diffusion coefficient of 10-6 cm2/sec, will diffuse approximately 105 Å in 1 sec. 105 Å (= 10-5 m = 10 mm) is approximately the diameter of an average human cell. Adapted from T. E. Creighton, Proteins, W.H.Freeman,1984. Length Dependence of the Radius of Gyration of Polypeptides Adapted from T. E. Creighton, Proteins W.H.Freeman, 1984 Adapted from T. E. Creighton, Proteins, W.H.Freeman,1984. Enough with the theory!! How do I purify a protein?