A suggestion for purifying the complex: let's say there is a 5mL gap between
the complex and one of its (smaller)constituents A. You can pre-load the
column with, say, 5mL of A at 1uM, then inject the complex at 80-100uM, to
be injected right after the pre-load. This should provide approximately
equilibrium conditions, so that the complex should be basically 1:1 when it
comes out, even with a high Koff. (Alternatively, for true equilibrium
conditions, just equilibrate the entire column in A, then inject the
complex.)
JPK
----- Original Message -----
From: "Justin Hall" <hallj...@onid.orst.edu>
To: <CCP4BB@JISCMAIL.AC.UK>
Sent: Friday, November 19, 2010 7:32 AM
Subject: Re: [ccp4bb] relationship between B factors and Koff
Hi Sebastiano,
I have had some experience with protein:protein complexes with KD ~
10-1 uM, kinetic characterization and trying to purify a complex of
these proteins using SEC. While I would say that if you have reliable
evidence from SPR that you have a fast on (high Kon), then you must
have a fast off (high Koff) because by definition KD = 10 E-6 =
Koff/Kon. However, I have observed several systems where you have a KD
~ 10-1 uM, but the kinetics are not fast on/fast off. In my
experience, I have never seen anything in the crystal structures of
the weak affinity complexes I have solved that would coorelate
B-factors to Kon/Koff, and while it might be tempting for you to draw
this comparison in your structure, I would warn that this is too large
a leap without further (non-anecdotal) evidence.
As a further note, during SEC purification of complexes, I have
observed that you generally have to have the complexes at at least 5
to 10-fold higher initial concentration if you want to purify the
complex, which you are only pushing with your 80-100 uM high end
concentration. A colleague of mine once told me this is due to a 5 to
10-fold dilution effect upon addition to the column, but I have never
verified this nor read any primary source that validated this so I
cannot supply a reference (others might be able to help here). Good
luck and cheers~
~Justin
Quoting Sebastiano Pasqualato <sebastiano.pasqual...@ifom-ieo-campus.it>:
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
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