Biochemistry 412

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Biochemistry 412 Analytical Preparative
Protein Chemistry I 1 February 2005
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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)
gtgtgt 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 gt 9
Adapted from T. E. Creighton, Proteins W.H.Freema
n, 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
are still 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 into the 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 from T. E. Creighton, Proteins W.H.Freema
n, 1984
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ltr2gt1/2 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. ltRGgt2 ltr2gt/6 for large polymers.
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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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.
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Some Examples of Diffusion Coefficients
Adapted from T. E. Creighton, Proteins,
W.H.Freeman,1984.
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Therefore, an average, garden-variety
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.
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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 from T. E. Creighton, Proteins W.H.Freema
n, 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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