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Biochemistry 412
Analytical Protein ChemistrySome Biophysical Properties of Proteins
6 February 2007
Positively-charged basic residues (K, R, & H)
Negatively-charged acidic residues (E & D)
Hydrophobic “patch”
Ligand binding pocket(active site)
ca. 40 ÅMacromolecular
dimensions:
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.
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.
s = MD(1-vρ)/RT
Remember:
Size and shape (reflected in the frictionalcoefficient) are the two things that usually mostaffect the hydrodynamic properties ofmacromolecules (btw, this is not limited toproteins -- applies to DNA and RNA, too).
Early centrifuges were massive affairs….
Modern ultracentrifuge
“A standard 20-pound fixed chamber ultracentrifugerotor (at 55,000 rpm) holds over a million joules ofenergy, which is roughly equivalent to the energyreleased by exploding several 1” sticks of dynamite.”
Source: UC BerkeleyEnvironment, Health and Safety Information Fact Sheet
Catastrophic failure of a rotor at MIT in 1999
http://web.mit.edu/charliew/www/centrifuge.html
Cornell University
http://www.ehs.cornell.edu/lrs/Centrifuge/CentrifugeDamages.htm
Translational Diffusion of Macromolecules
Adapted from T. E. Creighton, Proteins, W.H.Freeman,1984.
(5-20)
Q: can anyone guesswhy people werecelebrating aboutthis in 2005?
Some Examples of Diffusion Coefficients
Adapted from T. E. Creighton, Proteins, W.H.Freeman,1984.
Therefore, an average, garden-varietyprotein with a diffusion coefficient of 10-6
cm2/sec, will diffuse approximately:
105 Å (= 10-5 m = 10 µm) in 1 sec.or
104 Å (= 10-6 m = 1 µm) in 1 msec.
10 µm is approximately the diameter ofan average human cell, and 1 µm isapproximately the diameter of a cell nucleus.
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
Intrinsic viscosity is not often easy tomeasure!
Light scattering is a more usefulmethod to measure the hydrodynamicradius of a protein. And it can alsobe used to determine whether theprotein is aggregated or not.
Saridakis et al (2002) Acta Cryst. D58, 1597.
Dynamic light scattering enables you to follow protein aggregation in real time.
This web site gives a tutorial on light scattering:
http://www.brainshark.com/brainshark/vu/view.asp?pi=96364
[Note: light scattering measurements are sometimes nowcombined with gel filtration elution time measurements inthe same instrument to give an even more sophisticatedmeasurement of a protein’s effective hydrodynamic radius.]
Enough with the theory!!
How do I purify a protein?