Dear Professor Ethan,
I apologize, for the partial
information provided by me in the second email. For the *in vivo* assembled
(as-purified) protein, we have confirmed the [4Fe-4S] cluster by CD and
EPR. From the 415/280 ratio and Ferrozine assay, we have confirmed there is
one [4Fe-4S] cluster / per three monomers.
So, we anticipate the [4Fe-4S] cluster between 4
cysteines out of 6 cysteines form the helices-1 of three monomers. There
are also few examples in the PDB, for homo-trimeric proteins having only
one [4Fe-4S] cluster. Yet so far, I have not found any example with
different oligomeric states for the *in vivo* assembled (as-purified)
protein VS the *in vitro* reconstituted one.
Regards,
Bhanu
On Mon, Mar 19, 2018 at 11:56 PM, Ethan A Merritt <merr...@u.washington.edu>
wrote:
> 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
>
>
--
B4U
