Biochemistry 4122004

February 17th Lecture

Analytical & Preparative Protein Chemistry I

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.

Amino Acid Side Chains that are Negatively Charged

At neutral pH:

At pH > 9:

Adapted fromT. E. Creighton, ProteinsW.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 arestill 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 intothe 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 fromT. E. Creighton, ProteinsW.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.

Translational Diffusion of Macromolecules

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

(5-20)

Some Examples of Diffusion Coefficients

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

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

Length Dependence of the Radius of Gyration of Polypeptides

Adapted fromT. E. Creighton, ProteinsW.H.Freeman,1984

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

Enough with the theory!!

How do I purify a protein?

27

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