Sebastiano, We observed similar phenomenon in our protein-protein complex. This reference will give you details (Crystal Structure of an Electron Transfer Complex between Aromatic Amine Dehydrogenase- Azurin from Alcaligenes faecalis, Biochemistry, 45, 13500-13510, 2006).
Sukumar Narayanasami Sukumar NE-CAT, Building 436E Advanced Photon Source Argonne National Laboratory 9700 S.Cass Ave Argonne, IL 60439-4800 USA Tel: 630-252-0681 Fax: 630-252-0687 e-mail: [email protected] On Fri, Nov 19, 2010 at 6:58 AM, Sebastiano Pasqualato < [email protected]> 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 >
