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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 [[log in to unmask]] On Behalf Of Jacob Keller [[log in to unmask]] Sent: Monday, December 06, 2010 12:15 PM To: [log in to unmask] 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 <[log in to unmask]<mailto:[log in to unmask]>> 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: [log in to unmask]<mailto:[log in to unmask]> -- ******************************************* Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: [log in to unmask]<mailto:[log in to unmask]> *******************************************