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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 > >