[Myotox] Manuscript?

Fri Sep 2 06:04:03 MDT 2022

Hi Borries,
I agree with your assessment, it sounds like a good plan. I am on vacation
now, I will be back on September 19. If I can help in any way, please let
me know.
Best regards,
AnaG

Em qui., 1 de set. de 2022 às 11:37, Borries Demeler <demeler at gmail.com>
escreveu:

> Thank you Francisco and Sebastien for your helpful explanations, this
> makes a lot of sense.
> We will focus then on the NMR and the AUC results for this paper, and
> hopefully find some additional angle by which we can explore the higher
> order oligomers. I will send a first draft out soon.
> -Borries
>
> On Thu, Sep 1, 2022 at 7:04 AM Francisco Gomes-Neto <gomes.netof at gmail.com>
> wrote:
>
>>
>> Hi Borries, Bruno, and all!
>>
>>
>>
>> After our last meeting, We performed MD simulations starting from
>> randomly positioned Myotoxin and SDS molecules in a simulation box,
>> according to the experimental conditions. We knew that would be like find
>> out a needle in a haystack due to sampling limitations. In the end, we
>> detected dynamic changing aggregates; however, nothing similar to a
>> myotoxin hexamer. Without any experimental structural data, this MD
>> simulation setup generally is not well accepted and is considered highly
>> speculative.
>>
>>                 As Borries stated, the SDS concentration is below the
>> CMC. Without a relatively stable micelle surface, the hexamer formation
>> would depend on multiple and simultaneous interactions of single SDS
>> molecules and an entire population of Myotoxin molecules. It is an exciting
>> problem that must be accessed using a different approach.
>>
>>
>>
>> For now, unfortunately, I have no good news.
>>
>>
>>
>> Best regards
>>
>> Francisco
>>
>> Em qui., 1 de set. de 2022 às 00:23, Sebastien Poget <
>> Sebastien.Poget at csi.cuny.edu> escreveu:
>>
>>> Hi Borries and all,
>>>
>>> You indeed have a nice story that is coming together. Unfortunately, you
>>> are right that the electrophysiology experiments did not show any
>>> membrane-disrupting activity, although I was not able to do an exhaustive
>>> screening of conditions at that time. I actually have a new undergrad in my
>>> lab who expressed interest in the project, and a little bit of toxin left,
>>> so we could try again maybe with different SDS concentrations or other
>>> fatty acids/lysolipids. But I would certainly not hold back on publishing
>>> with what you have now. If we get the electrophysiology to work, it could
>>> be something useful to have in hand to address potential reviewer
>>> questions, or it could be part of a future paper.
>>>
>>> Best,
>>>
>>> Sebastien
>>>
>>>
>>>
>>> *From:* Myotox <myotox-bounces at biophysics.uleth.ca> * On Behalf Of *Borries
>>> Demeler
>>> *Sent:* Monday, August 22, 2022 2:26 PM
>>> *To:* Myotoxin-II discussion <myotox at biophysics.uleth.ca>
>>> *Subject:* Re: [Myotox] Manuscript?
>>>
>>>
>>>
>>> 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
>>>
>>> _______________________________________________
>>> Myotox mailing list
>>> Myotox at biophysics.uleth.ca
>>> https://demeler7.uleth.ca/mailman/listinfo/myotox
>>>
>>
>>
>> --
>> --
>> Francisco Gomes Neto
>> Pesquisador Assistente
>> Laboratório de Toxinologia
>> Instituto Oswaldo Cruz
>> Pavilhão Ozório de Almeida, Sala 5
>> Fundação Oswaldo Cruz-FIOCRUZ
>> Ave. Brasil, 4365 - Manguinhos
>> Caixa Postal 926
>> CEP 21040-900
>> Rio de Janeiro, RJ, Brasil.
>> Tel.: (55-21) 2562-1263
>> Fax : (55-21) Lab. 2562-1410
>>
>> _______________________________________________
>> Myotox mailing list
>> Myotox at biophysics.uleth.ca
>> https://demeler7.uleth.ca/mailman/listinfo/myotox
>>
> _______________________________________________
> Myotox mailing list
> Myotox at biophysics.uleth.ca
> https://demeler7.uleth.ca/mailman/listinfo/myotox
>

-- 
Ana Gisele da C. Neves Ferreira
Pesquisadora Titular em Saúde Pública
Chefe do Laboratorio de Toxinologia
Instituto Oswaldo Cruz - Fiocruz
Pavilhao Ozorio de Almeida, sala 05
Av. Brasil, 4365 - Manguinhos
Rio de Janeiro - RJ
21040-900 Brasil
(+5521) 2562-1381 / 98801-5726
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