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 <[log in to unmask]>:
> 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
>
>
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