Re: [ccp4bb] pH dependent conformational change
Hi Intrinsic fluorescence may be used to monitor such change qualitatively. Another option is red-edge excitation flourescence technique may work if there are suitable fluorophores (W or F) on the each of the two domains changing solvent accessibility upon rigid body motion. Amit. On Mon, Dec 6, 2010 at 11:56 PM, Jacob Keller j-kell...@fsm.northwestern.edu wrote: Well, I just got word that the protein is ~100kD anyway, so I think the HSQC is out the window anyway! Jacob On Mon, Dec 6, 2010 at 12:16 PM, Roopa Thapar rtha...@hwi.buffalo.edu wrote: I agree that the experiment is a good one and can easily be done, but without assignments I think the interpretation could be ambiguous. pH dependent chemical shift perturbations could occur far removed from the linker (either due to a conformational change or the change in chemical environment around the amide nucleus) and without any information about which residues are shifting it may be difficult to conclude that these perturbations are due to a change in domain orientation rather than other subtle pH dependent effects. If there are no perturbations, then of course one can conclude little or no conformational changes occur. The magnitude of the perturbation would depend on how extensive the conformational change is. One could specifically label the protein with 15N-labeled amino acids that are particularly unique to the linker - this would simplify the spectrum and the data may be easier to interpret. It is a good experiment to try however. Roopa From: Jacob Keller [j-kell...@fsm.northwestern.edu] Sent: Monday, December 06, 2010 12:54 PM To: Roopa Thapar Cc: CCP4BB@jiscmail.ac.uk Subject: Re: [ccp4bb] pH dependent conformational change Even without assignments, wouldn't a dramatic shift be seen in the interacting residues? Also, I suggested the method because it is pretty easy, probably doable in a week... Jacob On Mon, Dec 6, 2010 at 11:24 AM, Roopa Thapar rtha...@hwi.buffalo.edu wrote: If there are backbone NMR assignments available then, definately a pH titration using HSQCs would give site specific information. These are easy experiments if someone can help you set them up. The perturbations should map to the inter-domain interface. If there are no assignments for the protein, spectral changes in response to pH would be harder to interpret. You could try FRET by introducing two probes - one in each domain. Roopa From: CCP4 bulletin board [ccp...@jiscmail.ac.uk] On Behalf Of Jacob Keller [j-kell...@fsm.northwestern.edu] Sent: Monday, December 06, 2010 12:15 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] pH dependent conformational change Wouldn't a HSQC of 15N-labeled protein be a relatively easy yes/no experiment? Maybe it would not be incredibly definitive? Jacob On Mon, Dec 6, 2010 at 11:10 AM, Mischa Machius mach...@med.unc.edu mailto:mach...@med.unc.edu wrote: Daniel, You'll probably have to monitor pH changes through size changes of your protein, provided the structural changes will indeed cause size changes. You said easy, so that probably rules out Small-Angle X-Ray Scattering (SAXS), but that would be the highest-resolution method. You can try static and dynamic light scattering, analytical ultracentrifugation and fluorescence anisotropy. If you are really lucky, size exclusion chromatography might work too. And then there are the difficult ways... MM On Dec 6, 2010, at 11:59 AM, Daniel Jin wrote: Dear CCP4 colleagues, We have a protein that is composed of two domains connected by a short peptide linker. We have some indirect evidence showing that the two domains may somehow move against each other when exposed to different pH. It is unlikely to have any obvious secondary structure change since each domain behaves like a rigid body. I am wondering whether there is any “easy” way, biochemically or biophysically, to monitor the conformational changes in solution. Many thanks. As far as I know most of the pH sensing stories are linked to histidine residue. Can you point me to any references that show a different pH sensing mechanism (other than His)? Thanks. Best, Daniel --- Mischa Machius, PhD Director, Center for Structural Biology Assoc. Professor, Dept. of Pharmacology Member, Lineberger Comprehensive Cancer Center University of North Carolina 4079 Genetic Medicine CB#7365 120 Mason Farm Road Chapel Hill, NC 27599-7365, U.S.A. tel: +1-919-843-4485 fax: +1-919-966-5640 email: mach...@unc.edumailto:mach...@med.unc.edu -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training
Re: [ccp4bb] pH dependent conformational change
I would like to point out that HSQC could still be applied even in such a large protein. TROSY-HSQC has been successful in improving peaks in spectra of large protein. Typically the sample would need to be deuterated to see the full effect of TROSY, but even a partial deuteration can improve signal. We have run TROSY-spectra of a complex (75kDa) with no deuteration and that spectrum is much better than a normal HSQC spectrum. Dan
[ccp4bb] pH dependent conformational change
Dear CCP4 colleagues, We have a protein that is composed of two domains connected by a short peptide linker. We have some indirect evidence showing that the two domains may somehow move against each other when exposed to different pH. It is unlikely to have any obvious secondary structure change since each domain behaves like a rigid body. I am wondering whether there is any “easy” way, biochemically or biophysically, to monitor the conformational changes in solution. Many thanks. As far as I know most of the pH sensing stories are linked to histidine residue. Can you point me to any references that show a different pH sensing mechanism (other than His)? Thanks. Best, Daniel
Re: [ccp4bb] pH dependent conformational change
Daniel, You'll probably have to monitor pH changes through size changes of your protein, provided the structural changes will indeed cause size changes. You said easy, so that probably rules out Small-Angle X-Ray Scattering (SAXS), but that would be the highest-resolution method. You can try static and dynamic light scattering, analytical ultracentrifugation and fluorescence anisotropy. If you are really lucky, size exclusion chromatography might work too. And then there are the difficult ways... MM On Dec 6, 2010, at 11:59 AM, Daniel Jin wrote: Dear CCP4 colleagues, We have a protein that is composed of two domains connected by a short peptide linker. We have some indirect evidence showing that the two domains may somehow move against each other when exposed to different pH. It is unlikely to have any obvious secondary structure change since each domain behaves like a rigid body. I am wondering whether there is any “easy” way, biochemically or biophysically, to monitor the conformational changes in solution. Many thanks. As far as I know most of the pH sensing stories are linked to histidine residue. Can you point me to any references that show a different pH sensing mechanism (other than His)? Thanks. Best, Daniel --- Mischa Machius, PhD Director, Center for Structural Biology Assoc. Professor, Dept. of Pharmacology Member, Lineberger Comprehensive Cancer Center University of North Carolina 4079 Genetic Medicine CB#7365 120 Mason Farm Road Chapel Hill, NC 27599-7365, U.S.A. tel: +1-919-843-4485 fax: +1-919-966-5640 email: mach...@unc.edu
Re: [ccp4bb] pH dependent conformational change
Wouldn't a HSQC of 15N-labeled protein be a relatively easy yes/no experiment? Maybe it would not be incredibly definitive? Jacob On Mon, Dec 6, 2010 at 11:10 AM, Mischa Machius mach...@med.unc.edu wrote: Daniel, You'll probably have to monitor pH changes through size changes of your protein, provided the structural changes will indeed cause size changes. You said easy, so that probably rules out Small-Angle X-Ray Scattering (SAXS), but that would be the highest-resolution method. You can try static and dynamic light scattering, analytical ultracentrifugation and fluorescence anisotropy. If you are really lucky, size exclusion chromatography might work too. And then there are the difficult ways... MM On Dec 6, 2010, at 11:59 AM, Daniel Jin wrote: Dear CCP4 colleagues, We have a protein that is composed of two domains connected by a short peptide linker. We have some indirect evidence showing that the two domains may somehow move against each other when exposed to different pH. It is unlikely to have any obvious secondary structure change since each domain behaves like a rigid body. I am wondering whether there is any “easy” way, biochemically or biophysically, to monitor the conformational changes in solution. Many thanks. As far as I know most of the pH sensing stories are linked to histidine residue. Can you point me to any references that show a different pH sensing mechanism (other than His)? Thanks. Best, Daniel --- Mischa Machius, PhD Director, Center for Structural Biology Assoc. Professor, Dept. of Pharmacology Member, Lineberger Comprehensive Cancer Center University of North Carolina 4079 Genetic Medicine CB#7365 120 Mason Farm Road Chapel Hill, NC 27599-7365, U.S.A. tel: +1-919-843-4485 fax: +1-919-966-5640 email: mach...@unc.edu mach...@med.unc.edu -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] pH dependent conformational change
Hi SAXS can be a right tool. However, how big is short peptide linker? Check Nature paper by Askarieh G. and Hedhammar M. for non-His pH sensor. cheers Alex Am 06.12.2010 um 17:59 schrieb Daniel Jin jin_xiec...@yahoo.com: Dear CCP4 colleagues, We have a protein that is composed of two domains connected by a short peptide linker. We have some indirect evidence showing that the two domains may somehow move against each other when exposed to different pH. It is unlikely to have any obvious secondary structure change since each domain behaves like a rigid body. I am wondering whether there is any “easy” way, biochemically or biophy sically, to monitor the conformational changes in solution. Many t hanks. As far as I know most of the pH sensing stories are linked to histidine residue. Can you point me to any references that show a different pH sensing mechanism (other than His)? Thanks. Best, Daniel
Re: [ccp4bb] pH dependent conformational change
If there are backbone NMR assignments available then, definately a pH titration using HSQCs would give site specific information. These are easy experiments if someone can help you set them up. The perturbations should map to the inter-domain interface. If there are no assignments for the protein, spectral changes in response to pH would be harder to interpret. You could try FRET by introducing two probes - one in each domain. Roopa From: CCP4 bulletin board [ccp...@jiscmail.ac.uk] On Behalf Of Jacob Keller [j-kell...@fsm.northwestern.edu] Sent: Monday, December 06, 2010 12:15 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] pH dependent conformational change Wouldn't a HSQC of 15N-labeled protein be a relatively easy yes/no experiment? Maybe it would not be incredibly definitive? Jacob On Mon, Dec 6, 2010 at 11:10 AM, Mischa Machius mach...@med.unc.edumailto:mach...@med.unc.edu wrote: Daniel, You'll probably have to monitor pH changes through size changes of your protein, provided the structural changes will indeed cause size changes. You said easy, so that probably rules out Small-Angle X-Ray Scattering (SAXS), but that would be the highest-resolution method. You can try static and dynamic light scattering, analytical ultracentrifugation and fluorescence anisotropy. If you are really lucky, size exclusion chromatography might work too. And then there are the difficult ways... MM On Dec 6, 2010, at 11:59 AM, Daniel Jin wrote: Dear CCP4 colleagues, We have a protein that is composed of two domains connected by a short peptide linker. We have some indirect evidence showing that the two domains may somehow move against each other when exposed to different pH. It is unlikely to have any obvious secondary structure change since each domain behaves like a rigid body. I am wondering whether there is any “easy” way, biochemically or biophysically, to monitor the conformational changes in solution. Many thanks. As far as I know most of the pH sensing stories are linked to histidine residue. Can you point me to any references that show a different pH sensing mechanism (other than His)? Thanks. Best, Daniel --- Mischa Machius, PhD Director, Center for Structural Biology Assoc. Professor, Dept. of Pharmacology Member, Lineberger Comprehensive Cancer Center University of North Carolina 4079 Genetic Medicine CB#7365 120 Mason Farm Road Chapel Hill, NC 27599-7365, U.S.A. tel: +1-919-843-4485 fax: +1-919-966-5640 email: mach...@unc.edumailto:mach...@med.unc.edu -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edumailto:j-kell...@northwestern.edu ***
Re: [ccp4bb] pH dependent conformational change
Even without assignments, wouldn't a dramatic shift be seen in the interacting residues? Also, I suggested the method because it is pretty easy, probably doable in a week... Jacob On Mon, Dec 6, 2010 at 11:24 AM, Roopa Thapar rtha...@hwi.buffalo.edu wrote: If there are backbone NMR assignments available then, definately a pH titration using HSQCs would give site specific information. These are easy experiments if someone can help you set them up. The perturbations should map to the inter-domain interface. If there are no assignments for the protein, spectral changes in response to pH would be harder to interpret. You could try FRET by introducing two probes - one in each domain. Roopa From: CCP4 bulletin board [ccp...@jiscmail.ac.uk] On Behalf Of Jacob Keller [j-kell...@fsm.northwestern.edu] Sent: Monday, December 06, 2010 12:15 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] pH dependent conformational change Wouldn't a HSQC of 15N-labeled protein be a relatively easy yes/no experiment? Maybe it would not be incredibly definitive? Jacob On Mon, Dec 6, 2010 at 11:10 AM, Mischa Machius mach...@med.unc.edumailto:mach...@med.unc.edu wrote: Daniel, You'll probably have to monitor pH changes through size changes of your protein, provided the structural changes will indeed cause size changes. You said easy, so that probably rules out Small-Angle X-Ray Scattering (SAXS), but that would be the highest-resolution method. You can try static and dynamic light scattering, analytical ultracentrifugation and fluorescence anisotropy. If you are really lucky, size exclusion chromatography might work too. And then there are the difficult ways... MM On Dec 6, 2010, at 11:59 AM, Daniel Jin wrote: Dear CCP4 colleagues, We have a protein that is composed of two domains connected by a short peptide linker. We have some indirect evidence showing that the two domains may somehow move against each other when exposed to different pH. It is unlikely to have any obvious secondary structure change since each domain behaves like a rigid body. I am wondering whether there is any “easy” way, biochemically or biophysically, to monitor the conformational changes in solution. Many thanks. As far as I know most of the pH sensing stories are linked to histidine residue. Can you point me to any references that show a different pH sensing mechanism (other than His)? Thanks. Best, Daniel --- Mischa Machius, PhD Director, Center for Structural Biology Assoc. Professor, Dept. of Pharmacology Member, Lineberger Comprehensive Cancer Center University of North Carolina 4079 Genetic Medicine CB#7365 120 Mason Farm Road Chapel Hill, NC 27599-7365, U.S.A. tel: +1-919-843-4485 fax: +1-919-966-5640 email: mach...@unc.edumailto:mach...@med.unc.edu -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edumailto:j-kell...@northwestern.edu *** -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] pH dependent conformational change
I agree that the experiment is a good one and can easily be done, but without assignments I think the interpretation could be ambiguous. pH dependent chemical shift perturbations could occur far removed from the linker (either due to a conformational change or the change in chemical environment around the amide nucleus) and without any information about which residues are shifting it may be difficult to conclude that these perturbations are due to a change in domain orientation rather than other subtle pH dependent effects. If there are no perturbations, then of course one can conclude little or no conformational changes occur. The magnitude of the perturbation would depend on how extensive the conformational change is. One could specifically label the protein with 15N-labeled amino acids that are particularly unique to the linker - this would simplify the spectrum and the data may be easier to interpret. It is a good experiment to try however. Roopa From: Jacob Keller [j-kell...@fsm.northwestern.edu] Sent: Monday, December 06, 2010 12:54 PM To: Roopa Thapar Cc: CCP4BB@jiscmail.ac.uk Subject: Re: [ccp4bb] pH dependent conformational change Even without assignments, wouldn't a dramatic shift be seen in the interacting residues? Also, I suggested the method because it is pretty easy, probably doable in a week... Jacob On Mon, Dec 6, 2010 at 11:24 AM, Roopa Thapar rtha...@hwi.buffalo.edu wrote: If there are backbone NMR assignments available then, definately a pH titration using HSQCs would give site specific information. These are easy experiments if someone can help you set them up. The perturbations should map to the inter-domain interface. If there are no assignments for the protein, spectral changes in response to pH would be harder to interpret. You could try FRET by introducing two probes - one in each domain. Roopa From: CCP4 bulletin board [ccp...@jiscmail.ac.uk] On Behalf Of Jacob Keller [j-kell...@fsm.northwestern.edu] Sent: Monday, December 06, 2010 12:15 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] pH dependent conformational change Wouldn't a HSQC of 15N-labeled protein be a relatively easy yes/no experiment? Maybe it would not be incredibly definitive? Jacob On Mon, Dec 6, 2010 at 11:10 AM, Mischa Machius mach...@med.unc.edumailto:mach...@med.unc.edu wrote: Daniel, You'll probably have to monitor pH changes through size changes of your protein, provided the structural changes will indeed cause size changes. You said easy, so that probably rules out Small-Angle X-Ray Scattering (SAXS), but that would be the highest-resolution method. You can try static and dynamic light scattering, analytical ultracentrifugation and fluorescence anisotropy. If you are really lucky, size exclusion chromatography might work too. And then there are the difficult ways... MM On Dec 6, 2010, at 11:59 AM, Daniel Jin wrote: Dear CCP4 colleagues, We have a protein that is composed of two domains connected by a short peptide linker. We have some indirect evidence showing that the two domains may somehow move against each other when exposed to different pH. It is unlikely to have any obvious secondary structure change since each domain behaves like a rigid body. I am wondering whether there is any “easy” way, biochemically or biophysically, to monitor the conformational changes in solution. Many thanks. As far as I know most of the pH sensing stories are linked to histidine residue. Can you point me to any references that show a different pH sensing mechanism (other than His)? Thanks. Best, Daniel --- Mischa Machius, PhD Director, Center for Structural Biology Assoc. Professor, Dept. of Pharmacology Member, Lineberger Comprehensive Cancer Center University of North Carolina 4079 Genetic Medicine CB#7365 120 Mason Farm Road Chapel Hill, NC 27599-7365, U.S.A. tel: +1-919-843-4485 fax: +1-919-966-5640 email: mach...@unc.edumailto:mach...@med.unc.edu -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edumailto:j-kell...@northwestern.edu *** -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] pH dependent conformational change
Well, I just got word that the protein is ~100kD anyway, so I think the HSQC is out the window anyway! Jacob On Mon, Dec 6, 2010 at 12:16 PM, Roopa Thapar rtha...@hwi.buffalo.edu wrote: I agree that the experiment is a good one and can easily be done, but without assignments I think the interpretation could be ambiguous. pH dependent chemical shift perturbations could occur far removed from the linker (either due to a conformational change or the change in chemical environment around the amide nucleus) and without any information about which residues are shifting it may be difficult to conclude that these perturbations are due to a change in domain orientation rather than other subtle pH dependent effects. If there are no perturbations, then of course one can conclude little or no conformational changes occur. The magnitude of the perturbation would depend on how extensive the conformational change is. One could specifically label the protein with 15N-labeled amino acids that are particularly unique to the linker - this would simplify the spectrum and the data may be easier to interpret. It is a good experiment to try however. Roopa From: Jacob Keller [j-kell...@fsm.northwestern.edu] Sent: Monday, December 06, 2010 12:54 PM To: Roopa Thapar Cc: CCP4BB@jiscmail.ac.uk Subject: Re: [ccp4bb] pH dependent conformational change Even without assignments, wouldn't a dramatic shift be seen in the interacting residues? Also, I suggested the method because it is pretty easy, probably doable in a week... Jacob On Mon, Dec 6, 2010 at 11:24 AM, Roopa Thapar rtha...@hwi.buffalo.edu wrote: If there are backbone NMR assignments available then, definately a pH titration using HSQCs would give site specific information. These are easy experiments if someone can help you set them up. The perturbations should map to the inter-domain interface. If there are no assignments for the protein, spectral changes in response to pH would be harder to interpret. You could try FRET by introducing two probes - one in each domain. Roopa From: CCP4 bulletin board [ccp...@jiscmail.ac.uk] On Behalf Of Jacob Keller [j-kell...@fsm.northwestern.edu] Sent: Monday, December 06, 2010 12:15 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] pH dependent conformational change Wouldn't a HSQC of 15N-labeled protein be a relatively easy yes/no experiment? Maybe it would not be incredibly definitive? Jacob On Mon, Dec 6, 2010 at 11:10 AM, Mischa Machius mach...@med.unc.edumailto:mach...@med.unc.edu wrote: Daniel, You'll probably have to monitor pH changes through size changes of your protein, provided the structural changes will indeed cause size changes. You said easy, so that probably rules out Small-Angle X-Ray Scattering (SAXS), but that would be the highest-resolution method. You can try static and dynamic light scattering, analytical ultracentrifugation and fluorescence anisotropy. If you are really lucky, size exclusion chromatography might work too. And then there are the difficult ways... MM On Dec 6, 2010, at 11:59 AM, Daniel Jin wrote: Dear CCP4 colleagues, We have a protein that is composed of two domains connected by a short peptide linker. We have some indirect evidence showing that the two domains may somehow move against each other when exposed to different pH. It is unlikely to have any obvious secondary structure change since each domain behaves like a rigid body. I am wondering whether there is any “easy” way, biochemically or biophysically, to monitor the conformational changes in solution. Many thanks. As far as I know most of the pH sensing stories are linked to histidine residue. Can you point me to any references that show a different pH sensing mechanism (other than His)? Thanks. Best, Daniel --- Mischa Machius, PhD Director, Center for Structural Biology Assoc. Professor, Dept. of Pharmacology Member, Lineberger Comprehensive Cancer Center University of North Carolina 4079 Genetic Medicine CB#7365 120 Mason Farm Road Chapel Hill, NC 27599-7365, U.S.A. tel: +1-919-843-4485 fax: +1-919-966-5640 email: mach...@unc.edumailto:mach...@med.unc.edu -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edumailto:j-kell...@northwestern.edu *** -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu *** -- *** Jacob Pearson Keller Northwestern
