Biochemistry 412 2004 February 17th Lecture Analytical & Preparative Protein Chemistry I
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Transcript of Biochemistry 412 2004 February 17th Lecture Analytical & Preparative Protein Chemistry I
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Biochemistry 4122004
February 17th Lecture
Analytical & Preparative Protein Chemistry I
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Positively-charged basic residues (K, R, & H)
Negatively-charged acidic residues (E & D)
Hydrophobic “patch”
Ligand binding pocket(active site)
ca. 40 Å
Macromoleculardimensions:
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.
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Amino Acid Side Chains that are Negatively Charged
At neutral pH:
At pH > 9:
Adapted fromT. E. Creighton, ProteinsW.H.Freeman,1984
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Amino Acid Side Chains that are Positively Charged
At neutral pH:
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Water forms a hydration shell around proteins.
The properties of this bound water arestill the subject of many experimental
and theoretical investigations.
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Makarov et al (1998) Biopolymers 45, 469.
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Makarov et al (2000) Biophys. J. 76, 2966.
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Makarov et al (2002) Acc. Chem. Res. 35, 376.
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Purification schemes vary, depending on the source of the protein
and its intrinsic biophysical properties...
…some flow-charts for typical schemes follow.
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Purification Scheme for Proteins from their Natural Source
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Purification Scheme for Soluble Recombinant Proteins
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Purification Scheme for Insoluble Recombinant Proteins
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Purification Scheme for Membrane-Associated Proteins
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But first some theory….
We need to delve a bit more deeply intothe hydrodynamic properties of proteins so that
you understand why things work the way they do
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Adapted from T. E. Creighton, Proteins, W.H.Freeman,1984.
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Adapted fromT. E. Creighton, ProteinsW.H.Freeman,1984
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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.
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Adapted from T. E. Creighton, Proteins, W.H.Freeman,1984.
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Translational Diffusion of Macromolecules
Adapted from T. E. Creighton, Proteins, W.H.Freeman,1984.
(5-20)
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Some Examples of Diffusion Coefficients
Adapted from T. E. Creighton, Proteins, W.H.Freeman,1984.
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Adapted from T. E. Creighton, Proteins, W.H.Freeman,1984.
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Length Dependence of the Radius of Gyration of Polypeptides
Adapted fromT. E. Creighton, ProteinsW.H.Freeman,1984
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Adapted from T. E. Creighton, Proteins, W.H.Freeman,1984.
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Enough with the theory!!
How do I purify a protein?
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ChromatographyLiquid flow
Liquid flow
4:37990909
Time 1 2 3 4 5
Separation according to: -molecular weight/ size-charge-hydrophobicity-affinity
Sample containing proteins or peptides
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