Yes, this is where I heard of the technique, and my suggestion was a
modification of that, but my references were:
Hummel JP & Dreyer WJ (1962) Measurement of protein-binding phenomena by gel
filtration. Biochim Biophys Acta 63:530-532.
Ratner D (1974) The interaction bacterial and phage proteins with
immobilized Escherichia coli RNA polymerase. J Mol Biol 88(2):373-383.
JPK
----- Original Message -----
From: "Tanner, John J." <tanne...@missouri.edu>
To: <CCP4BB@JISCMAIL.AC.UK>
Sent: Friday, November 19, 2010 11:25 AM
Subject: Re: [ccp4bb] relationship between B factors and Koff
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"
<sebastiano.pasqual...@ifom-ieo-campus.it> 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 resonance).
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,
ciao
s
--
Sebastiano Pasqualato, PhD
IFOM-IEO Campus
Dipartimento di Oncologia Sperimentale
Istituto Europeo di Oncologia
via Adamello, 16
20139 - Milano
Italy
tel +39 02 9437 5094
fax +39 02 9437 5990
*******************************************
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
Dallos Laboratory
F. Searle 1-240
2240 Campus Drive
Evanston IL 60208
lab: 847.491.2438
cel: 773.608.9185
email: j-kell...@northwestern.edu
*******************************************
