[Myotox] Manuscript?

[Borries Demeler]

Hi Bruno,
I think the gel at different SDS concentrations is a great idea! Is this
something you could do? I would love to see the result. I recall that Amy
had to really fine-tune the ratio of SDS to protein in order to get the
oligomerization to a stable hexamer to work. My biggest concern however
would be that the protein species that are formed are not really
encapsulated in an SDS micelle at the lower concentrations, so their gel
migration pattern may be different from the typical 1% SDS PAGE. But it
never hurts to try, and if you see something bigger than a dimer that would
already be quite important. My guess is that the SDS concentration for the
hexamer formation is so low that we don't have any micelles, and SDS
doesn't form a regular micelle but acts more like glue to tie the
individual monomers together. That's just a simplistic idea, I don't have
any data to support that view.

Regarding the MD simulations, I'll refer to the expert, Francisco, but I
would love to see if there is a stable hexamer or heptamer configuration
that could be shown as a model for the oligomerization we saw. Maybe some
of the structural details determined by Paul could help inform the initial
model to try MD with?
-Borries




On Sun, Aug 21, 2022 at 10:19 PM Bruno Lomonte <bruno.lomonte at ucr.ac.cr>
wrote:

> Dear Borries and all,
>
> the data and conclusions reached thus far seem quite impressive to me,
> so I tend to believe this would be a good moment to put it together in a
> report for EBJ as suggested
>
> I would only have two very naive questions to you experts on these
> biophysical aspects:
>
> 1 - would it be worth to see the behavior of Mt-II in a series of
> SDS-PAGE gels having the same final SDS concentrations as those tested
> in the AUC experiments? for example by casting a gel with several
> spacers and run side-by-side the Mt-II varying only the SDS? I am not
> sure of what will happen or how useful the results would be, it is
> mostly a curiosity for visualizing what would happen as predicted by the
> AUC studies
>
> 2 - in the simulations by Francisco, would it be of interest to also try
> the same in silico experiment using the coordinates of Mt-II reported in
> the suramin complex (which was reported to have a different dimeric
> organization), 1Y4L, I would guess eliminating the suramin from the
> data, to see what is the outcome and compare with that of 1CLP? again, I
> am not sure if this could provide any useful information to this part of
> the study, just a curiosity because it could be that the 1CLP crystal
> dimer perhaps can be interpreted alternatively in its dimerization as a
> "compact" instead of the "extended" structure (as proposed by Fontes'
> group) - would that influence the outcome?
>
> best regards to all, and thank you Borries for nicely integrating all
> that has been done until now!
>
> Bruno
>
> +++++++++++++++++++++++
>
>
> On 8/21/2022 5:37 PM, Borries Demeler wrote:
> > Dear Colleagues,
> > it's been quiet on this list, and I am wondering if we reached a state
> where we want to publish the results we have collected so far for the
> mytoxin-II story. Allow me to provide a brief summary of our observations:
> >
> > 1. By AUC, MT-II remains monomeric in PBS up to very high concentrations
> (I believe 20 mg/ml was the highest we studied),
> > 2. At high [SDS] (>CMC), we observed a structure that could be a dimer.
> This is replicated in SDS-PAGE and in X-ray crystallography
> > 3. In AUC, we observed formation of a discrete hexamer or heptamer of
> MT-II in the presence of very low SDS.
> > 4. Increasing [SDS] would result in higher order aggregates/oligomers
> than hexamer, but s-value distributions were not discrete and quite
> heterogeneous, and finally dissociate into apparent dimer species.
> > 5. The same effect could not be reproduced with other lipids or
> detergents
> > 6. NMR shows an unusually strong interaction between MT-II and SDS at
> lower SDS concentrations. The alpha methylene exhibits high stress similar
> to that seen in an epoxy ring. Ar first sight it appears to be an AB
> quartet. Simulations show that the 2JHH of the methylene is very small
> indicating strain.
> > 7. electrophysiology experiments with bilayer membranes did not produce
> results - Sebastien, are there any updates?
> > 8. negative staining and cryoEM turned out to be a dead end
> > 9. MD simulations involving SDS by Francisco suggest a dimer formation
> >
> > I would like to know what, if any, experiments should be performed
> before we decide to publish? I propose to write up what we have so far and
> send it to Eur. Biophysical Journal. We are editing a special issue to
> celebrate the 25th AUC conference anniversary, held this past July in
> Lethbridge. If you are interested in contributing to this article, please
> indicate what data/methods you would like to contribute. Since the major
> discovery here is based on AUC (hexamer formation), I would propose that we
> submit this as an AUC focused manuscript.
> >
> > I'm open to any and all suggestions and would like to get your feedback.
> >
> > I'm also attaching a summary of what we have from Francisco and from our
> lab.
> >
> > Looking forward to get your feedback.
> >
> > Regards, -Borries
> >
> > _______________________________________________
> > Myotox mailing list
> > Myotox at biophysics.uleth.ca
> > https://demeler7.uleth.ca/mailman/listinfo/myotox
>
> --
> Bruno Lomonte, Ph.D.
> Instituto Clodomiro Picado
> Facultad de Microbiología
> Universidad de Costa Rica
> San José, COSTA RICA
>
> tel.of. (+506) 2511 7888
> bruno.lomonte at ucr.ac.cr
>
>
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