Dear Bernhard I share your intuition: we should expect to observe different shapes of titration curves depending on whether A or B2 is in the syringe (titration and reverse titration). I suppose that you want to test your hypothesis that, eventually, you get A2B2. Independently of any kinetic considerations , one should consider the possibility that the successive equilibria A+B2<-->AB2 and A + AB2 <-->A2B2 have distinct Kds and distinct DeltaH (which would of course be more favorable to detect these two distinct binding events, if they exist). I therefore suggest that you perform both possible titrations (if each protein can be sufficiently concentrated to be in the syringe) and that you try to fit each titration curve with the same model of interaction. If this works well, then the must would be to fit both titration curves at the same time with the same model of interaction . This is certainly the most demanding method to test a hypothesis and this is not at all equivalent to fit independently the two kinds of titration curves as you can imagine. (Let's say that this would amount to refine a single molecular model against crystal data from two space groups).The problem is of the practical possibility of doing such a joint fit with the available programs. I personally do such things with my own (not user-friendly!) programs. As far a I know, this is not possible, neither with Origin or PEAQ from Malvern, nor with NanoAnalyze from TA. I know that AFFINImeter is quite flexible to allow using specific models, but I'm not sure it would allow you to make such a global fit. I don't know about the possibility of SEDPHAT developed by P. Schuck. I hope this fits with your expectations. Best Philippe Dumas
De: "Bernhard Rupp" <hofkristall...@gmail.com> À: CCP4BB@JISCMAIL.AC.UK Envoyé: Jeudi 3 Octobre 2019 17:05:56 Objet: [ccp4bb] ITC question -dimer vs monomer Hi Fellows, please let me ask the respective experts an ITC question: I have 2 proteins, stable and dialyzed in identical buffer. A is a monomer and B an obligate dimer. I suspect that eventually a A2B2 dimer will form. Intuitively, it should make a difference whether I titrate the dimer with the monomer or vice versa. In the first case, a momomer would initially meet a lot of free dimers, and I would expect that randomly, a AB2 complex is more likely to form than a A2B2 (let’s disregard any more complex colligative/cooperative effects). If I drip the dimer into the monomer pool, it is quite likely that the B dimer meets 2 free As, and I get right away a higher population of A2B2s. Maybe at dilutions of ITC and with sufficient equilibration that is not an issue at all (again, absent any cooperative effects that might alter the first Kd vs. the second, despite the sites on the dimer are at least initially equivalent). Can someone guide me towards literature about this or perhaps share some first-hand experience? Many thanks, BR ------------------------------------------------------ Bernhard Rupp [ http://www.hofkristallamt.org/ | http://www.hofkristallamt.org/ ] [ mailto:b...@hofkristallamt.org | b...@hofkristallamt.org ] +1 925 209 7429 +43 676 571 0536 ------------------------------------------------------ Many plausible ideas vanish at the presence of thought ------------------------------------------------------ To unsubscribe from the CCP4BB list, click the following link: [ https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 | https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 ] ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1
