Hi Gergő, Thank you for discussing it in detail. It is a great help. Based on the curve fit, we also believe that the two proteins bind independently to the DNA duplex with the same affinity. The DNA indeed contains 2 sites for protein binding. As far as the dimerization of protein is concerned, we have already confirmed, through different approaches, that the protein doesn't behave as a dimer in the solution.
Regards Abhishek *Dr. Abhishek Suman* *Ph.D (Structural Biology)* Indian Institute of Technology Hyderabad Kandi 502 284 Sangareddy Telangana INDIA Contact: +91 91002 74548, +91 80843 11898 Email: [email protected] ---------------------------------------------------- P* Please don't print this e-mail or attachment unless necessary. Preserve trees on the planet.* On Wed, Aug 17, 2022 at 12:24 AM Gergő Gógl <[email protected]> wrote: > Dear Abhishek, > > When you use a function with "one set of sites" and an “independent” > model, you most likely used some sort of quadratic binding formula. In case > the true binding equation is like you wrote [2A + B <-> (A2)B], then it > is not expected to look like a quadratic, but rather like a cubic formula. > > {Just solve this: Kd=( ( Atot - 0.5*[(A2)B] )^2 * (Btot - [(A2)B]) ) / ( [ > (A2)B] ) and you will see that the resulting function is cubic for the > concentration of the complex ([(A2)B]).} > > If you indeed had a perfect sigmoidal observation with no systematic > divergence from the fitted quadratic curve, I would not find any strong > indication that the binding function should be considered as cubic. > Instead, I would assume that: > > -the protein binding to the DNA follows a simple bimolecular binding model > and the DNA just happens to be able to bind two molecules of proteins > independently with the same affinity (within the detection thresholds of > the assay). [A + B_site1 <-> AB_site1 and A + B_site2 <-> AB_site2] > -or the protein binds as a stable dimer to the DNA molecule and therefore > the binding can be considered as pseudo bimolecular. (DNA+dimer <->complex) > In this case, you should change the protein concentration to "protein dimer > concentration" during fitting. [(A2) + B <-> (A2)B] > > In either case, your dissociation constant has a dimension of M and not > M^2. > > If your binding model really follows the reaction you proposed and you do > not like the first option where the affinities of the sites are identical, > you should use a "two independent set of sites" model. In this case, you > will end up with 2 independent affinities that are not identical and their > dimension will be in M. (If they are close, you are better if you use a one > site and assume that the sites are not distinguishable.) > > If your sites are not independent and you have strong cooperativity, you > will need to use more complex biochemistry (such as sequential binding) and > your Kd dimension may be different, but this is most likely not the case > because you had a decent fit with a simple. > > I hope I could help and did not miss something important. Please let me > know if others propose something different! > > Best, > Gergo > > ABHISHEK SUMAN <[email protected]> ezt írta > (időpont: 2022. aug. 16., K, 19:40): > >> Hello everyone! >> >> >> >> Hope this email finds you well. I have an off-topic question regarding >> ITC binding studies, which was asked by a reviewer. >> >> >> >> We performed an ITC binding study (using Affinity ITC, TA Instruments) to >> evaluate protein-DNA interaction which resulted in a perfect sigmoidal >> curve. We used the ‘one set of sites’ binding algorithm (“independent” >> model) for curve fitting and to calculate binding and thermodynamic >> parameters. The study suggested two copies of the protein binding to a >> single duplex DNA, i.e., the stoichiometry of protein:DNA is 2:1 (N=2). The >> ITC calculated the KD (equilibrium molar dissociation constant) in μM >> (micromolar). But the reviewer is asking to report the KD in >> (micromolar)^2 instead of micromolar mentioning that the binding reaction >> is 2A + B <-> (A2)B and the complex is (A2)B and not AB. Though we're >> trying to explain to the reviewer that we couldn't find any software that >> can compute the KD in (micromolar)^2 for the stoichiometry of 2 but he >> is not agreeing to it. We have used the NanoAnalyze software from the TA >> instrument. This software does not have a model to measure the KD in >> (micromolar)^2. >> >> >> I would be grateful if you could help me to resolve this problem or at >> least let me know what explanation might be appropriate to answer the >> reviewer’s concern that it’s a general practice to report the KD in the >> Molar irrespective of stoichiometry. >> >> >> >> Thanks in advance. >> >> >> >> Regards >> >> Abhishek >> >> >> *Dr. Abhishek Suman* >> >> *Ph.D (Structural Biology)* >> >> Indian Institute of Technology Hyderabad >> >> Kandi 502 284 Sangareddy >> >> Telangana INDIA >> >> Contact: +91 91002 74548, +91 80843 11898 >> >> Email: [email protected] >> >> ---------------------------------------------------- >> >> P* Please don't print this e-mail or attachment unless necessary. >> Preserve trees on the planet.* >> >> Disclaimer:- This footer text is to convey that this email is sent by >> one of the users of IITH. 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