Thanks to All for the extended & informative responses. If true thermodynamic equilibria
are realized, then I would agree that regardless of the pathway the endpoint (or integrated H) should be the same. The actual pathway and cooperativity probably will make this an interesting problem. I may keep bugging selected victims off-board once I have the first data. Many thanks again, BR From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> On Behalf Of Barone, Matthias Sent: Thursday, October 3, 2019 18:59 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] ITC question -dimer vs monomer As Reza already pointed out, ITC cannot tell you anything about the sub-processes that underlay an equilibrium, be it complicated like 2A+B2 <-> AB2 + A <-> A2B2, or with (virtually) no intermediate 2A+B2 <-> A2B2 due to a fast second forward reaction , or a simple one-to-one model A+B2 <-> AB2. Given the lack of additional information, its probably good to assume a simple one-to-one model and titrate either partner to the other. If you have two separate binding steps (with similar Kd for B2 for A as well as AB2 for A), you would measure an apparent affinity and would see the stochiometrics according to the inflection point (be it around equimolar excess or at 0.5 or 2, depending on whether you titrate A or B). If the reaction is more complicated and the the affinities for B2 for A differ significantly much from the affinity of AB2 for A, then a simple one-to-one would leave some notable information in the residual standard deviations (meaning, the residuals would not spread normally around the Regression line, but should show a wavy pattern). Sorry for the long mail.. Matthias Dr. Matthias Barone AG Kuehne, Rational Drug Design Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP) Robert-Rössle-Strasse 10 13125 Berlin Germany Phone: +49 (0)30 94793-284 future. From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK <mailto:CCP4BB@JISCMAIL.AC.UK> > On Behalf Of Bernhard Rupp Sent: Thursday, October 3, 2019 11:06 AM To: CCP4BB@JISCMAIL.AC.UK <mailto:CCP4BB@JISCMAIL.AC.UK> Subject: [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 (lets 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/ <https://urldefense.com/v3/__http:/www.hofkristallamt.org/__;!oCotSwSxbw8!SE 9mT6grUy1tHFSKLCraXt4bhlDri03OEMEyqQUCLAVSLsg3vwn0GTQtxbStgtNvxBs$> b...@hofkristallamt.org <mailto:b...@hofkristallamt.org> +1 925 209 7429 +43 676 571 0536 ------------------------------------------------------ Many plausible ideas vanish at the presence of thought ------------------------------------------------------ _____ To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB <https://urldefense.com/v3/__https:/www.jiscmail.ac.uk/cgi-bin/webadmin?SUBE D1=CCP4BB&A=1__;!oCotSwSxbw8!SE9mT6grUy1tHFSKLCraXt4bhlDri03OEMEyqQUCLAVSLsg 3vwn0GTQtxbStkIPgsQE$> &A=1 _____ To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB <https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1> &A=1 _____ To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB <https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1> &A=1 _____ To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB <https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1> &A=1 _____ To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB <https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1> &A=1 ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1
