On Monday, March 19, 2018 2:20:50 PM PDT PULSARSTRIAN wrote:
> Dear Professor Diana,
>
> Thank you very much for your
> comment, and we will certainly look at, if ‘domain swapping’ is any reason
> for the trimeric nature of the protein.
>
> There are two cysteines in helix-1 and two cysteines
> in helix 3. From our mutational studies (C to S), we confirmed in the *in
> vivo *assembled protein, that only helix-1 (the first two cysteines) form
> the Fe-S cluster. So, its altogether a completely new structure compared to
> reconstituted protein.
Your description of the in vivo characterization seems to be inconsistent.
1) 4Fe-4S cluster formed in vivo from 2 copies of helix-1, each contributing 2
Cys.
2) resulting in a trimer
How would that work?
Or is it that you are proposing that there is a single 4Fe/4S center per trimer,
formed stochastically from 4 of the 6 available Cys residues?
You don't mention having EPR for the in vivo assembly.
Have you determined the Fe/S stoichiometry in the trimer?
Ethan
>
> Regards,
>
> Bhanu
>
>
>
> On Mon, Mar 19, 2018 at 10:13 PM, Diana Tomchick <
> diana.tomch...@utsouthwestern.edu> wrote:
>
> > Is it possible that you have a case of domain swapping that causes the
> > trimeric assembly?
> >
> > Diana
> >
> > **************************************************
> > Diana R. Tomchick
> > Professor
> > Departments of Biophysics and Biochemistry
> > University of Texas Southwestern Medical Center
> > 5323 Harry Hines Blvd
> > <https://maps.google.com/?q=5323+Harry+Hines+Blvd&entry=gmail&source=g>.
> > Rm. ND10.214A
> > Dallas, TX 75390-8816
> > diana.tomch...@utsouthwestern.edu
> > (214) 645-6383 (phone)
> > (214) 645-6353 (fax)
> >
> > On Mar 19, 2018, at 3:07 PM, PULSARSTRIAN <bhanu.hydpri...@gmail.com>
> > wrote:
> >
> > Dear all,
> >
> > Sorry for the slightly off-topic question.
> >
> > I am working on a non-native, *de novo* [4Fe-4S]
> > protein, designed as a four-helix bundle. The * in vitro* reconstituted
> > protein assembles with [4Fe-4S] (confirmed by EPR) and exists in
> > monomer-dimer configuration (confirmed by SEC). These results have been
> > already published.
> >
> > Recently we could get the [4Fe-4S] assembly directly from
> > the *E. coli* (*in vivo* assembly). Everything is as expected (compared
> > to reconstituted protein), except the oligomerization state. The protein
> > assembles as trimer, in contrast to monomer-dimer configuration of the
> > reconstituted protein. The trimeric nature of the *in vivo* assembled
> > protein has been confirmed by SEC, SEC-SLS and SAXS.
> >
> >
> > *So, my question is, has anyone encountered such situation,
> > where the As-purified Fe-S protein having a completely different oligomeric
> > state compared to the in vitro reconstitution protein? *
> >
> >
> > Looking forward to hearing for some examples and/or references.
> >
> >
> > Regards,
> >
> > Bhanu
> >
> >
> > ------------------------------
> >
> > UT Southwestern
> >
> > Medical Center
> >
> > The future of medicine, today.
> >
>
>
>
>
--
Ethan A Merritt
Biomolecular Structure Center, K-428 Health Sciences Bldg
MS 357742, University of Washington, Seattle 98195-7742
