Re: [ccp4bb] ITC with heterogeneous protein
Dear Sajid, one first problem in your study is how-to adress if the deltaH mesured is caused by the ligand interaction, or by the modification of dimer-monomer equilibrium. You have to well caracterise your system dimer-monomer. One other problem is about the accessibility of the interaction site. If it's different between monomer and dimer, you have to know the ratio between these two state, to managed the proportion of active protein. Is it possible to separate the dimer from monomer ? Is this equilibrium concentration dependant ? In this case, you can try to execute your ITC experiment in low protein concentration. Or try to find condition in wich you can assume that you have large majority of dimer in your sample. To my mind it's difficult to start without more information. You can also try to titrate the protein with itself. If dimer/monomer is concentration dependent, you can try to titrate protein with itself. This can give you some information about the thermodynamics of the dimer/monomer formation. Hope to help. Nicolas De : CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] de la part de sajid akthar [b_sajid_...@yahoo.co.in] Envoyé : vendredi 18 juillet 2014 11:24 À : CCP4BB@JISCMAIL.AC.UK Objet : [ccp4bb] ITC with heterogeneous protein Dear All, This is an off-topic question. I have protein solution of heterogeneous (contains both monomer and dimer). I want to perform ITC with this protein. I doubt whether this heterogeneity will interfere the binding study. Any advice please. Thank you Sajid
Re: [ccp4bb] ITC with heterogeneous protein
Dear Sajid If the binding site of your ligand is remote from the dimerization interface, it should normally not be a problem. You will bind two ligands for a dimer and one ligand for a monomer and you should be able to fit the isotherm with one unique site even in presence of a mix of monomers and dimers. If the binding site is close to the dimerization interface or partially dissociate the dimer, this may give more complicate signals difficult to analyzed You can also try to evaluate with ITC or other biophysical technic, the Kd of your dimer. With ITC, the Kd of your dimer may be evaluated by injecting a concentrated solution of your protein via the serynge against a cell containing your dialysis buffer. You may observe an heat exchange due to dimer dissociation (see for example Li, J, Weis RM, 1996, with CheA) Knowing the Kd may help to design the ITC experiment. For example, you can use a concentration of protein 10 fold higher (if possible) than the Kd to have mainly dimers Cheers JB JB Charbonnier Laboratory of Structural Biology and Radiobiology iBiTec-S (Institute of Biology and Technologies of Saclay), CEA , FRANCE jb.charbonn...@cea.fr -Message d'origine- De : CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] De la part de sajid akthar Envoyé : vendredi 18 juillet 2014 11:24 À : CCP4BB@JISCMAIL.AC.UK Objet : [ccp4bb] ITC with heterogeneous protein Dear All, This is an off-topic question. I have protein solution of heterogeneous (contains both monomer and dimer). I want to perform ITC with this protein. I doubt whether this heterogeneity will interfere the binding study. Any advice please. Thank you Sajid
Re: [ccp4bb] ITC with heterogeneous protein
Hi Sajid, *Assuming* you have one site per monomer (rather than, say, one site per dimer), and *assuming* each binding event is completely independent ( I.e no co-operativity), you might just get away with running the experiment with the heterogeneous material. However, you might not be able to confidently make these assumptions, so imho it would be preferable to separate the monomer and dimer by SEC prior to ITC. If this is not possible, then pay close attention to the fit when you run the heterogeneous experiment. Poor fit to a one site model may indicate that these assumptions are invalid. Can you obtain stoichiometry information from a different technique? This might be very helpful. Hth, Dave Dr David C Briggs PhD http://about.me/david_briggs On 18 Jul 2014 10:25, sajid akthar b_sajid_...@yahoo.co.in wrote: Dear All, This is an off-topic question. I have protein solution of heterogeneous (contains both monomer and dimer). I want to perform ITC with this protein. I doubt whether this heterogeneity will interfere the binding study. Any advice please. Thank you Sajid
Re: [ccp4bb] ITC with heterogeneous protein
I have a similar case, where in there are multiple binding sites on the protein for the ligand and ligand induces dimerization.So it is not helpful even if I separate the monomer and dimer. If I titrate the dimer with ligand, the stoichiometry will completely change? Any suggestions will be helpful. On Fri, Jul 18, 2014 at 12:46 PM, David Briggs drdavidcbri...@gmail.com wrote: Hi Sajid, *Assuming* you have one site per monomer (rather than, say, one site per dimer), and *assuming* each binding event is completely independent ( I.e no co-operativity), you might just get away with running the experiment with the heterogeneous material. However, you might not be able to confidently make these assumptions, so imho it would be preferable to separate the monomer and dimer by SEC prior to ITC. If this is not possible, then pay close attention to the fit when you run the heterogeneous experiment. Poor fit to a one site model may indicate that these assumptions are invalid. Can you obtain stoichiometry information from a different technique? This might be very helpful. Hth, Dave Dr David C Briggs PhD http://about.me/david_briggs On 18 Jul 2014 10:25, sajid akthar b_sajid_...@yahoo.co.in wrote: Dear All, This is an off-topic question. I have protein solution of heterogeneous (contains both monomer and dimer). I want to perform ITC with this protein. I doubt whether this heterogeneity will interfere the binding study. Any advice please. Thank you Sajid
Re: [ccp4bb] ITC with heterogeneous protein
it depend of what you expected as information : In this case your measure is resulting from ligand binding AND dimerization (except if all the protein is already dimerized). I am not sur to understand, do you know how many binding sites exists on one monomer ? You should be able to determine the stoechiometry ligand/protein, but it's not necessarily the same between monomer and dimer. Is the ligand trigger for dimerization ? Or the dimerization appear in all case upon the concentration is sufficient ? De : CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] de la part de Ramesh V [ramesh.c...@gmail.com] Envoyé : vendredi 18 juillet 2014 14:42 À : CCP4BB@JISCMAIL.AC.UK Objet : Re: [ccp4bb] ITC with heterogeneous protein I have a similar case, where in there are multiple binding sites on the protein for the ligand and ligand induces dimerization.So it is not helpful even if I separate the monomer and dimer. If I titrate the dimer with ligand, the stoichiometry will completely change? Any suggestions will be helpful. On Fri, Jul 18, 2014 at 12:46 PM, David Briggs drdavidcbri...@gmail.commailto:drdavidcbri...@gmail.com wrote: Hi Sajid, *Assuming* you have one site per monomer (rather than, say, one site per dimer), and *assuming* each binding event is completely independent ( I.e no co-operativity), you might just get away with running the experiment with the heterogeneous material. However, you might not be able to confidently make these assumptions, so imho it would be preferable to separate the monomer and dimer by SEC prior to ITC. If this is not possible, then pay close attention to the fit when you run the heterogeneous experiment. Poor fit to a one site model may indicate that these assumptions are invalid. Can you obtain stoichiometry information from a different technique? This might be very helpful. Hth, Dave Dr David C Briggs PhD http://about.me/david_briggs On 18 Jul 2014 10:25, sajid akthar b_sajid_...@yahoo.co.inmailto:b_sajid_...@yahoo.co.in wrote: Dear All, This is an off-topic question. I have protein solution of heterogeneous (contains both monomer and dimer). I want to perform ITC with this protein. I doubt whether this heterogeneity will interfere the binding study. Any advice please. Thank you Sajid
Re: [ccp4bb] ITC with heterogeneous protein
If the following is not deleterious to your protein and its function you could introduce mutations that prevent dimerization. ~Jeff I have a similar case, where in there are multiple binding sites on the protein for the ligand and ligand induces dimerization.So it is not helpful even if I separate the monomer and dimer. If I titrate the dimer with ligand, the stoichiometry will completely change? Any suggestions will be helpful. On Fri, Jul 18, 2014 at 12:46 PM, David Briggs drdavidcbri...@gmail.com wrote: Hi Sajid, *Assuming* you have one site per monomer (rather than, say, one site per dimer), and *assuming* each binding event is completely independent ( I.e no co-operativity), you might just get away with running the experiment with the heterogeneous material. However, you might not be able to confidently make these assumptions, so imho it would be preferable to separate the monomer and dimer by SEC prior to ITC. If this is not possible, then pay close attention to the fit when you run the heterogeneous experiment. Poor fit to a one site model may indicate that these assumptions are invalid. Can you obtain stoichiometry information from a different technique? This might be very helpful. Hth, Dave Dr David C Briggs PhD http://about.me/david_briggs On 18 Jul 2014 10:25, sajid akthar b_sajid_...@yahoo.co.in wrote: Dear All, This is an off-topic question. I have protein solution of heterogeneous (contains both monomer and dimer). I want to perform ITC with this protein. I doubt whether this heterogeneity will interfere the binding study. Any advice please. Thank you Sajid
