Dera Careina going back to the original software question, I think you may be able to use the Rasmot-3D Pro server
http://biodev.cea.fr/rasmot3d/ Nucleic Acids Res. 2009 Jul;37(Web Server issue):W459-64. doi: 10.1093/nar/gkp304. Epub 2009 May 5. RASMOT-3D PRO: a 3D motif search webserver. Debret G, Martel A, Cuniasse P. Source Service d'Ingénierie Moléculaire des Protéines (SIMOPRO), iBiTec-S, DSV, CEA, CE-Saclay, 91191 Gif Sur Yvette Cedex, France. This allows you to upload coordinates containing a a motif (in your case two disulphide-bonded Cys residues from another protein), and the coordinates for a protein structure you wish to examine - in this case your dimeric coordinates. It will then search for pairs of CA-CB bonds in the protein that would be at the right relative angles and distances to form a disulphide if those residues were mutated to cysteines. There are a number of different conformations for disulphide bonds depending on the various torsion angles so you may need to choose a number of different search structures - though obviously 2-fold symmetric ones would be a good start. After your mutagenesis some of the suggestions that have already been made may allow you to oxidize them to a disulphide. best wishes Pete On 28 Feb 2013, at 14:52, Roger Rowlett wrote: > In the literature, you can find examples of air oxidation, oxidized > glutathione (alone), mixture of reduced and oxidized glutathione, and > hydrogen peroxide. The correct concentrations have to be found empirically. > We are just now mushing through this with an engineered disulfide variant. > Air oxidation partially worked for us--one of the engineered disulfides > worked, one did not. > > _______________________________________ > Roger S. Rowlett > Gordon & Dorothy Kline Professor > Department of Chemistry > Colgate University > 13 Oak Drive > Hamilton, NY 13346 > > tel: (315)-228-7245 > ofc: (315)-228-7395 > fax: (315)-228-7935 > email: rrowl...@colgate.edu > > On 2/28/2013 7:55 AM, Clemens Grimm wrote: >>> Along these lines, what reagents do people use to promote disuflide bonds, >>> i.e., the "anti-DTT?" >> >> Glutathione (red) + Glutathione (ox), redox potential is adjusted by varying >> the ratio. >> >> Best, >> Clemens >> >> >>> >>> JPK >>> >>> On Thu, Feb 28, 2013 at 2:06 AM, David Briggs >>> <drdavidcbri...@gmail.com>wrote: >>> >>>> You might want to try "Disulfide by design" >>>> >>>> http://cptweb.cpt.wayne.edu/DbD2/ >>>> >>>> Cheers >>>> >>>> Dave >>>> On Feb 28, 2013 6:55 AM, "Careina Edgooms" <careinaedgo...@yahoo.com> >>>> wrote: >>>> >>>>> Dear CCP4 members >>>>> >>>>> I wish to engineer a disulfide bond at the dimer interface of a protein I >>>>> am working with. Does anyone know of any available software to assist with >>>>> this? >>>>> >>>>> Best >>>>> Careina >>>>> >>>> >>> >>> >>> -- >>> ******************************************* >>> Jacob Pearson Keller, PhD >>> Postdoctoral Associate >>> HHMI Janelia Farms Research Campus >>> email: j-kell...@northwestern.edu >>> ******************************************* >>> >> >> >> >> -------------------------------------------------- >> Dr. Clemens Grimm >> Institut für Biochemie >> Biozentrum der Universität Würzburg >> Am Hubland >> D-97074 Würzburg >> Germany >> e-mail: clemens.gr...@biozentrum.uni-wuerzburg.de >> phone : +49 0931 31 84031 >> ------------------------------------------------- Prof Peter Artymiuk Krebs Institute Department of Molecular Biology & Biotechnology University of Sheffield Sheffield S10 2TN ENGLAND
