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 <[email protected]> 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 [[email protected]] > Sent: Monday, December 06, 2010 12:54 PM > To: Roopa Thapar > Cc: [email protected] > 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 <[email protected]> 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 [[email protected]] On Behalf Of Jacob Keller >> [[email protected]] >> Sent: Monday, December 06, 2010 12:15 PM >> To: [email protected] >> 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 >> <[email protected]<mailto:[email protected]>> 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: [email protected]<mailto:[email protected]> >> >> >> >> >> -- >> ******************************************* >> Jacob Pearson Keller >> Northwestern University >> Medical Scientist Training Program >> cel: 773.608.9185 >> email: [email protected]<mailto:[email protected]> >> ******************************************* >> > > > > -- > ******************************************* > Jacob Pearson Keller > Northwestern University > Medical Scientist Training Program > cel: 773.608.9185 > email: [email protected] > ******************************************* > -- ******************************************* Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: [email protected] *******************************************
