Hi Monika, Philippe is right, ITC can detect only binding with -dH > ~0.3 kcal/mole (depends on instrument). If the binding is Entropy driven, i.e. TdS >> -dH, than Kd might be quite low, like 20 uM, but ITC would still not detect it (this often happens in mutants). You can try to optimize experimental conditions, like reduce salt concentration in the buffer, change the buffer molecule (like replace HEPES/MES to PO4 or Tris), temperature, etc. In my experiments, HEPES/MES were often binding nonspecifically to ligand binding sites and reduced the heat...
Alex From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> on behalf of "DUMAS Philippe (IGBMC)" <p.du...@ibmc-cnrs.unistra.fr> Reply-To: "DUMAS Philippe (IGBMC)" <p.du...@ibmc-cnrs.unistra.fr> Date: Saturday, August 1, 2020 at 1:43 AM To: "CCP4BB@JISCMAIL.AC.UK" <CCP4BB@JISCMAIL.AC.UK> Subject: Re: [ccp4bb] Regarding difference in ITC and structure data [EXTERNAL EMAIL] 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" <chandravanshi.monik...@gmail.com> À: "CCP4BB" <CCP4BB@JISCMAIL.AC.UK> 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<https://nam03.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.jiscmail.ac.uk%2Fcgi-bin%2FWA-JISC.exe%3FSUBED1%3DCCP4BB%26A%3D1&data=02%7C01%7Caaleshin%40SBPDISCOVERY.ORG%7C9096a2d3d8324eccf9d908d835f6ed9a%7C0b162723004547deb0699f1a7aa955a1%7C0%7C1%7C637318681977036565&sdata=8ckrKWLNg9VToNhlcmLhJQhqLmN4TI0fjY%2B1lwphBeM%3D&reserved=0> ________________________________ To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1<https://nam03.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.jiscmail.ac.uk%2Fcgi-bin%2FWA-JISC.exe%3FSUBED1%3DCCP4BB%26A%3D1&data=02%7C01%7Caaleshin%40SBPDISCOVERY.ORG%7C9096a2d3d8324eccf9d908d835f6ed9a%7C0b162723004547deb0699f1a7aa955a1%7C0%7C1%7C637318681977046557&sdata=vYVXr778T2OLKSHjRTosQg99NNtczasjiutjWSqt2rA%3D&reserved=0> ######################################################################## 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/
