Re: [ccp4bb] ITC data clarification.
Hello, only a test of the biological function of mutants will tell whether your interface is an artefact or not. It is very well possible that an alanine mutations increases binding; you have to inspect the interface carefully whether there were for example buried hydrogen donors or flexible residues in the WT interface which made the interaction less than optimal. Regards Wim Dharma a écrit : Hello CCP4 users, Based on the crystal structure of a two molecule protein complex, I have mutated (alanine substitutions) one of the putative binding interface. The mutant binds with much higher affinity than the wild type. However, the signature plot of ITC data reveals a decrease in the enthalpy but increase on the entropy (deltaS). Thus overall increase in deltaG. I want to know if it’s relevant biological interface or a crystal artifact. Suggestions please. Thanks Regards Dharma Sent from my iPhone
Re: [ccp4bb] ITC data clarification.
hi dharma your ITC data suggest at least that your complex also exists in solution ITC never will answer to the question whether this is a “biological” interface one way getting around this, would be to check the response of your mutant versus WT in biological assay ALA substitutions soemtimes are not drastic enough to interrupt protein protein interactions introduction of a charged residue facing a hydrophobic pocket often is more efficient good luck Herman van Tilbeurgh Professor structural biology Directeur Adjoint Ecole Doctorale Innovation Thérapeutique: du fondamental à l'appliqué Institut de Biologie Intégrative de la Cellule - I2BC UMR 9198 CNRS- Université Paris Sud Team: Fonction et Architecture des Assemblages MacroMoléculaires http://www.i2bc.paris-saclay.fr/spip.php?article256 Batiment 430 91405 Orsay France Tel: 33 1 69 15 31 55 fax: 33 1 69 85 37 15 herman.van-tilbeu...@u-psud.fr > Le 8 déc. 2017 à 02:15, Dharma a écrit : > > Hello CCP4 users, > Based on the crystal structure of a two molecule protein complex, I have > mutated (alanine substitutions) one of the putative binding interface. The > mutant binds with much higher affinity than the wild type. > However, the signature plot of ITC data reveals a decrease in the enthalpy > but increase on the entropy (deltaS). Thus overall increase in deltaG. > I want to know if it’s relevant biological interface or a crystal artifact. > Suggestions please. > > Thanks > Regards > Dharma > Sent from my iPhone
[ccp4bb] ITC data clarification.
Hello CCP4 users, Based on the crystal structure of a two molecule protein complex, I have mutated (alanine substitutions) one of the putative binding interface. The mutant binds with much higher affinity than the wild type. However, the signature plot of ITC data reveals a decrease in the enthalpy but increase on the entropy (deltaS). Thus overall increase in deltaG. I want to know if it’s relevant biological interface or a crystal artifact. Suggestions please. Thanks Regards Dharma Sent from my iPhone
