Jan, I agree that the DTNB assay (Ellman's Reagent) can be used to quantify exposed Cysteines. Alternatively, you could do site directed mutagenesis and mutate significant amino acids responsible for the conformational change from the open to the closed state, with hopes to lock the protein in the closed conformation. However, this may prove to be tricky. If the protein is indeed an enzyme, you can also attempt to crystallize the protein with a transition state analog inhibitor, thus inhibiting turnover and locking the protein in the closed conformation.... Good luck!lorenzo
Lorenzo Ihsan FInci, Ph.D.Postdoctoral Scientist, Wang LaboratoryHarvard Medical SchoolDana-Farber Cancer InstituteBoston, MA Peking UniversityThe College of Life SciencesBeijing, China Date: Wed, 11 Jul 2012 11:12:17 -0400 From: kellydaugh...@gmail.com Subject: Re: [ccp4bb] Disulphide bonds and closed conformation To: CCP4BB@JISCMAIL.AC.UK Jan,If the Cys residues are accessible, you could try DTNB to quantify the number of Cys, thus determining if they are reduced or bridged. http://en.wikipedia.org/wiki/Ellman's_reagent Kelly******************************************************* Kelly Daughtry, Ph.D. Post-Doctoral Fellow, Raetz Lab Biochemistry Department Duke University Alex H. Sands, Jr. Building 303 Research Drive RM 250 Durham, NC 27710 P: 919-684-5178 ******************************************************* On Wed, Jul 11, 2012 at 11:08 AM, Jan Rashid Umar <jan...@googlemail.com> wrote: Dear all, I am working on a protein where I have to stabilize the closed conformation of the protein using disulphide bond. The strategy to design the cysteine mutants is based on the molecular dynamic simulations, and accordingly the residues were chosen. The ultimate goal is to trap the ligand in closed conformation of protein and crystallize it. I am facing few issues: Is there some reliable assay that can check the formation of disulphide bonds in protein. Additionally, does anybody knows another method(s) that can be used to trap a closed conformation. I look forward for your suggestions and discussions on this issue. Thanks very much! Jan
