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.edu<mailto:mach...@med.unc.edu> > >> > >> > >> > >> > >> -- > >> ******************************************* > >> Jacob Pearson Keller > >> Northwestern University > >> Medical Scientist Training Program > >> cel: 773.608.9185 > >> email: j-kell...@northwestern.edu<mailto: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 University > Medical Scientist Training Program > cel: 773.608.9185 > email: j-kell...@northwestern.edu > ******************************************* > -- ------------------------------------------------------------------- AMIT DAS PROTEIN CRYSTALLOGRAPHY SOLID STATE PHYSICS DIVISION BARC, TROMBAY, MUMBAI-400085 INDIA. Alt. E-mail:amit...@barc.gov.in <e-mail%3aamit...@barc.gov.in> PHONE:+91-22-25594688/4063 FAX:+91-22-25505151
