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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?