Hi Monika. Can you please give us some more information about the ITC experiments? Does the affinity change between maltose, maltotriose, maltotetraose and maltopentaose or do they all bind equally strong? And, does N stay at roughly equimolar ratios or is there a trend from 1 towards 0.2 for the wt protein (assuming your protein has one site to bind one titrand)? I have attached a pdf to make it a bit easier to show. It could either be that maltose binds much weaker than the pentamer or that the pentamer binds with 1:5 stoichiometry. In both cases you are able to get an affinity for the pentamer but not for the monomer, whose saturation curve would be too flat to fit. Do you see any dH for the maltose and maltotriose with the mutant, or is it just linear and not fittable like the red curve in the pdf? The reason that you see them however in the structure could be because of higher protein concentration and excess. Saturation depends on both variables. You use 100x molar excess in the crystallization droplet, and maybe a higher protein concentration to easily saturate a weak interaction of, say 5-8mM. In a crystal you got even higher protein concentration which would allow the mutant to bind maltose and maltotriose even better.
So, if you see indeed some dH released by the maltose binding to your mutant, I would first try to push the syringe and cell concentrations to the limit. If you see no dH change, follow Philippe's suggestion and changing the temperature would be a good idea. best, 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 ________________________________ From: CCP4 bulletin board <[email protected]> on behalf of DUMAS Philippe (IGBMC) <[email protected]> Sent: Saturday, August 1, 2020 10:42:16 AM To: [email protected] Subject: Re: [ccp4bb] Regarding difference in ITC and structure data Monika Did you try ITC experiments at different temperatures ? Delta H may be null, or close to zero, at some temperature without implying that there is no binding ! Philippe Dumas ________________________________ De: "monika chandravanshi" <[email protected]> À: "CCP4BB" <[email protected]> Envoyé: Samedi 1 Août 2020 09:57:24 Objet: [ccp4bb] Regarding difference in ITC and structure data Dear All, I am working on a carbohydrate-binding protein, which co-crystallizes with maltose, maltotriose, maltotetraose and maltopentaose and the same can be supported by ITC experiments as well. Also, the mutant protein (X2Y) co-crystallizes with maltose, maltotriose, maltotetraose and maltopentaose, however, the binding of only maltotriose and maltotetraose could be observed through ITC. For your information, the ITC conditions are the same for all the ligands and the ligand concentration used in ITC is same as used in crystallization (100x of protein concentrations). Moreover, from structural analysis, we have observed that the binding mode of all ligands is the same. I request your suggestion on why maltose and maltopentaose do not show any binding to the mutant protein in ITC experiments. Looking forward to suggestions. Best Regards, Monika ________________________________ To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 ________________________________ To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
ITC_vs_crystal.pdf
Description: ITC_vs_crystal.pdf
