Hi Todd, disulfides (both intra and intermolecular) are quite common in extremophile cytosolic proteins. I routinely come across these. Many of these disulfides are retained when the proteins are overexpressed in E.coli regardless of the strong reducing environment. Usually these disulfides are relatively buried and the attack by reducing molecules such as thioredoxin is hindered. Many of them are only partially reduced when mercaptoethanol or are reducing agents are used in purification and the structures have to be modelled with partial occupancies of reduced/oxidised disulfides. In such cases we are trying to avoid adding the reducing agents.
I have also seen many examples of mesophilic bacterial proteins and mammalian proteins forming disulfides when overexpressed in E.coli. Sometimes to get homogeneous sample we add disulfide enforcing agent (diamide) as early as induction stage when overexpressing in E.coli. Best wishes, Misha ________________________________________ From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Reza Khayat [rkha...@ccny.cuny.edu] Sent: 20 December 2014 19:05 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] non-specific disulfide bonds in crystal structures Hi Todd, A concern of mine is that you may be looking at an artifact of the domain. Whatever assays you decide to do, make sure that they are also performed for the full sized protein. Best wishes, Reza Reza Khayat, PhD Assistant Professor The City College of New York Department of Chemistry, MR-1135 160 Convent Avenue New York, NY 10031 Tel. (212) 650-6070 www.khayatlab.org ---- Original message ---- >Date: Sat, 20 Dec 2014 10:44:25 -0800 >From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> (on behalf of Christine Gee <chr...@gmail.com>) >Subject: Re: [ccp4bb] non-specific disulfide bonds in crystal structures >To: CCP4BB@JISCMAIL.AC.UK > > Hi Todd, > I used to work on PNMT which also is supposedly > monomeric and formed a disulfide between monomers in > the crystals. > See http://www.sciencedirect.com/science/article/pii/S157096390 5000968?via=ihub > We showed that it was irrelevant to activity. > Cheers > Christine > Sent from my iPad > On 20 Dec 2014, at 9:52 am, Todd Jason Green > <tgr...@uab.edu> wrote: > > Hello All- > > I have recently determined a domain structure of a > larger protein. The structure shows a clear > disulfide bond between two monomers in the > asymmetric unit. I'm trying to figure out if this > is an artifact of the crystal packing or has > biological relevance. The protein has been > reported to function as a monomer. If I look at > the pool of protein on a SDS-PAGE gel under > non-reducing conditions, I see that a smaller > percentage (~15-20%) of the protein runs as a > dimer. In the structure, the association has > 2-fold symmetry with about 29% of the monomeric > surface area buried between the dimer. Can anyone > point me in the direction of a paper describing a > non-specific disulfide in a crystal, or perhaps a > criteria for assessing specificity? I will do some > functional studies, but I'm looking for some info > on a lazy saturday. > > Thanks in advance. Best- > Todd
