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.
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.
>
--
B4U
