Biochemistry 412
Analytical Protein ChemistrySome Biophysical Properties of Proteins
15 February 2008
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
Water forms a hydration shell around proteins.
The properties of this bound water arestill the subject of many experimental
and theoretical investigations.
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.
<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.
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).
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 inch thick sticks ofdynamite.
Source: UC BerkeleyEnvironment, Health and Safety Information Fact Sheet
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?
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 10 msec.
10 µm is approximately the diameter ofan average human cell, and 1 µm isapproximately the diameter of a cell nucleus.
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.]