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] *******************************************
