[ccp4bb] phosphoprotein crystallization
I am trying to crystallize a protein that I am phosphorylating in-vitro. There is no way that I can purify the phosphorylated protein from unphosphorylated one. I tried Ion exchange and gel filtration for separating them and they are not working. Mass spec result shows the phosphorylation at desired site but in the crystal structure, I am not seeing density for phosphoryl group. I haven't use any phosphatase inhibitor during crystallization. is it possible that phosphoryl group is getting lost due to radiation damage or due to phosphatases, my protein is getting dephosphorylated before making crystal? I typically get crystals within 24 hours. if I am losing phosphoryl group due to radiation damage, should I still expect to see conformational changes due to phosphorylation? in phosphorylated protein, an alpha helix has to move to accommodate phosphoryl group but I am not seeing any change in that alpha helix. did any one tried to set crystal tray with phosphorylation reaction mix without further purification? thank you for suggestion. Dhiraj
Re: [ccp4bb] phosphoprotein crystallization
Thank you Prof Lewis. Its serine residue which is getting phosphorylated. the pI of my protein is around 8.2. So native gel ( around pH 8.3) is also not working for me. I tried ion exchange at pH 7.0, 7.5 and 8.5 but my protein is either coming in flow through or very poorly binding to Q and SP sepharose. does any one have any good suggestion for finding out phosphorylation status of a basic protein with pI around 8.0. So far we were using mass spec on tryptic digest but I need a simple and relatively quick method because its not practical to run mass spec on each and every fractions before pooling them. Thanks Dhiraj On Jan 19, 2015, at 11:13 AM, Rick Lewis r.le...@ncl.ac.uk wrote: is this a phosphorylation on serine, threonine or tyrosine? if so, they should be quite stable between ~pH 5 and 8. the MS results are probably not quantitative, just qualitative, and i would expect that the addition of a net negative 2 charge from the phosphoryl group should make a difference to your protein on ion exchange. i would run a non-denaturing PAGE, and look for differences in Rf for your protein. i bet that the phosphorylation has not gone anywhere near completion, especially since you seem to expect a conformational change to occur to accommodate the PO3, and this is not observed. if your preps are clean, you shouldn't need a p'tase inhibitor. good luck rick On 19/01/15 16:54, Dhiraj Srivastava wrote: I am trying to crystallize a protein that I am phosphorylating in-vitro. There is no way that I can purify the phosphorylated protein from unphosphorylated one. I tried Ion exchange and gel filtration for separating them and they are not working. Mass spec result shows the phosphorylation at desired site but in the crystal structure, I am not seeing density for phosphoryl group. I haven't use any phosphatase inhibitor during crystallization. is it possible that phosphoryl group is getting lost due to radiation damage or due to phosphatases, my protein is getting dephosphorylated before making crystal? I typically get crystals within 24 hours. if I am losing phosphoryl group due to radiation damage, should I still expect to see conformational changes due to phosphorylation? in phosphorylated protein, an alpha helix has to move to accommodate phosphoryl group but I am not seeing any change in that alpha helix. did any one tried to set crystal tray with phosphorylation reaction mix without further purification? thank you for suggestion. Dhiraj -- R. J. Lewis Professor of Structural Biology Institute for Cell and Molecular Biosciences Faculty of Medical Sciences Tel: +44 (0)191 208 5482 University of Newcastle Fax: +44 (0)191 208 7424 Newcastle upon Tyne, NE2 4HH, UKEmail: r.le...@ncl.ac.uk URL: sbl.ncl.ac.uk
Re: [ccp4bb] phosphoprotein crystallization
Try running a regular SDS page gel w/o boiling your sample. Some P proteins run differently on gel due to the conformational change that is induced. I agree that you probably have a mixture in which the P form might be the minor species. I'd probably try to get the IEX to work; given that the sample sticks poorly to the column one might expect the -2 introduced by the phosphate to make a big difference. Did you try loading the IEX column at low salt (maybe as low as 20 mM or so)? Bert From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Srivastava, Dhiraj [dhiraj-srivast...@uiowa.edu] Sent: Monday, January 19, 2015 5:39 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] phosphoprotein crystallization Thank you Prof Lewis. Its serine residue which is getting phosphorylated. the pI of my protein is around 8.2. So native gel ( around pH 8.3) is also not working for me. I tried ion exchange at pH 7.0, 7.5 and 8.5 but my protein is either coming in flow through or very poorly binding to Q and SP sepharose. does any one have any good suggestion for finding out phosphorylation status of a basic protein with pI around 8.0. So far we were using mass spec on tryptic digest but I need a simple and relatively quick method because its not practical to run mass spec on each and every fractions before pooling them. Thanks Dhiraj On Jan 19, 2015, at 11:13 AM, Rick Lewis r.le...@ncl.ac.uk wrote: is this a phosphorylation on serine, threonine or tyrosine? if so, they should be quite stable between ~pH 5 and 8. the MS results are probably not quantitative, just qualitative, and i would expect that the addition of a net negative 2 charge from the phosphoryl group should make a difference to your protein on ion exchange. i would run a non-denaturing PAGE, and look for differences in Rf for your protein. i bet that the phosphorylation has not gone anywhere near completion, especially since you seem to expect a conformational change to occur to accommodate the PO3, and this is not observed. if your preps are clean, you shouldn't need a p'tase inhibitor. good luck rick On 19/01/15 16:54, Dhiraj Srivastava wrote: I am trying to crystallize a protein that I am phosphorylating in-vitro. There is no way that I can purify the phosphorylated protein from unphosphorylated one. I tried Ion exchange and gel filtration for separating them and they are not working. Mass spec result shows the phosphorylation at desired site but in the crystal structure, I am not seeing density for phosphoryl group. I haven't use any phosphatase inhibitor during crystallization. is it possible that phosphoryl group is getting lost due to radiation damage or due to phosphatases, my protein is getting dephosphorylated before making crystal? I typically get crystals within 24 hours. if I am losing phosphoryl group due to radiation damage, should I still expect to see conformational changes due to phosphorylation? in phosphorylated protein, an alpha helix has to move to accommodate phosphoryl group but I am not seeing any change in that alpha helix. did any one tried to set crystal tray with phosphorylation reaction mix without further purification? thank you for suggestion. Dhiraj -- R. J. Lewis Professor of Structural Biology Institute for Cell and Molecular Biosciences Faculty of Medical Sciences Tel: +44 (0)191 208 5482 University of Newcastle Fax: +44 (0)191 208 7424 Newcastle upon Tyne, NE2 4HH, UKEmail: r.le...@ncl.ac.uk URL: sbl.ncl.ac.uk
Re: [ccp4bb] phosphoprotein crystallization
Hi, there: If you just want to know the percentage of phosphorylation, I highly recommend phospho-tag gel. You can buy the phospho-tag, and make gels your self. It's very effective. Shawn Toronto On 2015-01-19, at 1:01 PM, Bert Van-Den-Berg wrote: Try running a regular SDS page gel w/o boiling your sample. Some P proteins run differently on gel due to the conformational change that is induced. I agree that you probably have a mixture in which the P form might be the minor species. I'd probably try to get the IEX to work; given that the sample sticks poorly to the column one might expect the -2 introduced by the phosphate to make a big difference. Did you try loading the IEX column at low salt (maybe as low as 20 mM or so)? Bert From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Srivastava, Dhiraj [dhiraj-srivast...@uiowa.edu] Sent: Monday, January 19, 2015 5:39 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] phosphoprotein crystallization Thank you Prof Lewis. Its serine residue which is getting phosphorylated. the pI of my protein is around 8.2. So native gel ( around pH 8.3) is also not working for me. I tried ion exchange at pH 7.0, 7.5 and 8.5 but my protein is either coming in flow through or very poorly binding to Q and SP sepharose. does any one have any good suggestion for finding out phosphorylation status of a basic protein with pI around 8.0. So far we were using mass spec on tryptic digest but I need a simple and relatively quick method because its not practical to run mass spec on each and every fractions before pooling them. Thanks Dhiraj On Jan 19, 2015, at 11:13 AM, Rick Lewis r.le...@ncl.ac.uk wrote: is this a phosphorylation on serine, threonine or tyrosine? if so, they should be quite stable between ~pH 5 and 8. the MS results are probably not quantitative, just qualitative, and i would expect that the addition of a net negative 2 charge from the phosphoryl group should make a difference to your protein on ion exchange. i would run a non-denaturing PAGE, and look for differences in Rf for your protein. i bet that the phosphorylation has not gone anywhere near completion, especially since you seem to expect a conformational change to occur to accommodate the PO3, and this is not observed. if your preps are clean, you shouldn't need a p'tase inhibitor. good luck rick On 19/01/15 16:54, Dhiraj Srivastava wrote: I am trying to crystallize a protein that I am phosphorylating in-vitro. There is no way that I can purify the phosphorylated protein from unphosphorylated one. I tried Ion exchange and gel filtration for separating them and they are not working. Mass spec result shows the phosphorylation at desired site but in the crystal structure, I am not seeing density for phosphoryl group. I haven't use any phosphatase inhibitor during crystallization. is it possible that phosphoryl group is getting lost due to radiation damage or due to phosphatases, my protein is getting dephosphorylated before making crystal? I typically get crystals within 24 hours. if I am losing phosphoryl group due to radiation damage, should I still expect to see conformational changes due to phosphorylation? in phosphorylated protein, an alpha helix has to move to accommodate phosphoryl group but I am not seeing any change in that alpha helix. did any one tried to set crystal tray with phosphorylation reaction mix without further purification? thank you for suggestion. Dhiraj -- R. J. Lewis Professor of Structural Biology Institute for Cell and Molecular Biosciences Faculty of Medical Sciences Tel: +44 (0)191 208 5482 University of Newcastle Fax: +44 (0)191 208 7424 Newcastle upon Tyne, NE2 4HH, UKEmail: r.le...@ncl.ac.uk URL: sbl.ncl.ac.uk
Re: [ccp4bb] phosphoprotein crystallization
an effective modification to your protein preparations would be to add phosphatase inhibitor tablets during all stages of purification. Pierce sell a combination protease and PI tablet that is effective and economical to be added during protein preps. Also you invitro buffers should have inhibitors http://www.piercenet.com/product/pierce-protease-phosphatase-inhibitor-tablets P On Mon, Jan 19, 2015 at 8:54 AM, Dhiraj Srivastava dhiraj-srivast...@uiowa.edu wrote: I am trying to crystallize a protein that I am phosphorylating in-vitro. There is no way that I can purify the phosphorylated protein from unphosphorylated one. I tried Ion exchange and gel filtration for separating them and they are not working. Mass spec result shows the phosphorylation at desired site but in the crystal structure, I am not seeing density for phosphoryl group. I haven't use any phosphatase inhibitor during crystallization. is it possible that phosphoryl group is getting lost due to radiation damage or due to phosphatases, my protein is getting dephosphorylated before making crystal? I typically get crystals within 24 hours. if I am losing phosphoryl group due to radiation damage, should I still expect to see conformational changes due to phosphorylation? in phosphorylated protein, an alpha helix has to move to accommodate phosphoryl group but I am not seeing any change in that alpha helix. did any one tried to set crystal tray with phosphorylation reaction mix without further purification? thank you for suggestion. Dhiraj -- P
