This doesn't directly address your question, but since the subject of analyzing 
protein-protein interactions with gel filtration is raised on this bb 
occasionally, I thought I would mention that there are cases in which 
conventional gel filtration chromatography fails to provide evidence of a known 
protein:protein interaction.  In such cases, the Hummel-Dreyer gel filtration 
method is sometimes used.  It involves supplementing the running buffer with 
one of the proteins, so you need a lot of protein.  Here are two references:

Proc. Natl. Acad. Sci. USA 88 (1991)
Biochemisrry (1993) 32, 11124-11131

On 11/19/10 6:58 AM, "Sebastiano Pasqualato" 
<> wrote:

Hi all,
I have a crystallographical/biochemical problem, and maybe some of you guys can 
help me out.

We have recently crystallized a protein:protein complex, whose Kd has been 
measured being ca. 10 uM (both by fluorescence polarization and surface plasmon 
Despite the 'decent' affinity, we couldn't purify an homogeneous complex in 
size exclusion chromatography, even mixing the protein at concentrations up to 
80-100 uM each.
We explained this behavior by assuming that extremely high Kon/Koff values 
combine to give this 10 uM affinity, and the high Koff value would account for 
the dissociation going on during size exclusion chromatography. We have partial 
evidence for this from the SPR curves, although we haven't actually measured 
the Kon/Koff values.

We eventually managed to solve the crystal structure of the complex by mixing 
the two proteins (we had to add an excess of one of them to get good 
diffraction data).
Once solved the structure (which makes perfect biological sense and has been 
validated), we get mean B factors for one of the component (the larger) much 
lower than those of the other component (the smaller one, which we had in 
excess). We're talking about 48 Å^2 vs. 75 Å^2.

I was wondering if anybody has had some similar cases, or has any hint on the 
possible relationship it might (or might not) exist between high a Koff value 
and high B factors (a relationship we are tempted to draw).

Thanks in advance,
best regards,

Sebastiano Pasqualato, PhD
Dipartimento di Oncologia Sperimentale
Istituto Europeo di Oncologia
via Adamello, 16
20139 - Milano

tel +39 02 9437 5094
fax +39 02 9437 5990

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