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<div dir="auto">It is set to Zurich time.</div>
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<div class="x_gmail_quote">On Dec. 7, 2021 9:28 a.m., Borries Demeler <demeler@gmail.com> wrote:<br type="attribution">
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<div class="PlainText">Caution: This email was sent from someone outside of the University of Lethbridge. Do not click on links or open attachments unless you know they are safe. Suspicious emails should be forwarded to phishing@uleth.ca.<br>
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<br>
Dear Bruno,<br>
thanks for getting back - since it is essential that Bruno is part of<br>
this discussion I will try once more with a new poll for next week:<br>
<br>
<a href="https://doodle.com/poll/ut3psa4k68tiu6ve?utm_source=poll&utm_medium=link">https://doodle.com/poll/ut3psa4k68tiu6ve?utm_source=poll&utm_medium=link</a><br>
<br>
Please check your browsers, I believe the times listed are for Mountain<br>
time, for comparison, Mondays options are 11:00 am - 4:00 pm, 1 hour<br>
sections.<br>
<br>
Please all respond by the end of the day so we can release the other<br>
times for everyone. I will send out the consensus by tonight.<br>
<br>
Thanks, -Borries<br>
<br>
On Mon, Dec 06, 2021 at 10:27:49PM -0600, Bruno Lomonte wrote:<br>
> Dear Borries and All,<br>
><br>
> Many thanks Borries for envisioning all these interesting avenues to study<br>
> and better understand the toxin. It will be exciting to explore the<br>
> possibility of pore formation or other mechanisms related to myotoxicity.<br>
> There has been a large knowledge gap regarding the interaction of this type<br>
> of toxin with membranes, so this is much needed work!<br>
><br>
> I olny regret at this moment that the following days (proposed in the Doodle<br>
> poll) are fully booked in my case. Sorry! If there are other alternative<br>
> options for everyone to meet and discuss, please let us know.<br>
><br>
> Here attached is an old study done on liposome permeabilization (including<br>
> liposomes made of non-hydrolyzable ether lipids - the toxin can disrupt<br>
> them, but nothing further has been done along this line of work, as far as I<br>
> know.<br>
><br>
> Best regards,<br>
><br>
> Bruno<br>
><br>
> ++++++++++++++++++++++<br>
><br>
><br>
> On 12/6/2021 12:00 PM, Borries Demeler wrote:<br>
> > Hello everybody,<br>
> ><br>
> > it has been a while since we have reported back on our NMR findings,<br>
> > so it is about time to pick up this interesting project again and get<br>
> > everyone up-to-date on the latest status.<br>
> ><br>
> > If you recall, Amy found that by using significantly lower SDS<br>
> > concentrations than CMC that myotoxin-II formed a very stable and<br>
> > homogeneous oligomer with about 6 or 7 myotoxin-II molecules in it. When<br>
> > SDS concentration was increased, it was possible to disrupt this oligomer<br>
> > and generate increasing amounts of heterogeneous oligomeric forms, with<br>
> > the predominant form being dimer. This observation matched nicely with<br>
> > the SDS PAGE results reported earlier by Susumu Uchiyama's group.<br>
> ><br>
> > But of course, more interesting to all of us should be what the<br>
> > structure of the oligomeric species was, and if it potentially formed<br>
> > a membrane penetrating pore, or has some other functional relevance.<br>
> ><br>
> > If you recall, and in summary, Paul and his group has investigated the<br>
> > interaction of SDS with myotoxin-II, and after multiple iterations of<br>
> > different ratios of SDS to myotoxin-II, concluded the following:<br>
> ><br>
> > "There seems to be an unusually strong interaction with SDS. The<br>
> > alpha methylene exhibits high stress similar to that seen in an epoxy<br>
> > ring. Ar first sight it appears to be an AB quartet. Simulations show<br>
> > that the 2JHH of the methylene is very small indicating strain."<br>
> ><br>
> > Paul also mentioned that the NMR studies are hindered by the large<br>
> > background signals and really would require labeling to allow more<br>
> > sophisticated multi-dimensional analysis.<br>
> ><br>
> > I also pursued the potential to get more info with cryo-EM by sending<br>
> > the samples to the University of Manitoba for study by Pauline Padilla<br>
> > and Joerg Stetefeld, but this angle unfortunately did not go anywhere,<br>
> > since negative staining screens did not produce the desired results,<br>
> > suggesting cryoEM is out of the question.<br>
> ><br>
> > Separately, I contacted Bruce Bowler at the University of Montana, who<br>
> > studies protein-membrane interactions, and he offered an array of other<br>
> > lipids he had used for crystallizing cytochrome-C interacting with<br>
> > membranes and still has in the fridge. So we could try to use some of<br>
> > these lipids to replicate Amy's results with other lipid systems.<br>
> ><br>
> > Recently, I heard a very interesting talk by Sebastien Poget on the<br>
> > interactions of various animal toxins with lipids, so I shared our<br>
> > findings with him. He wrote the following:<br>
> ><br>
> > "Thanks for your e-mails, it looks like you have a very interesting<br>
> > toxin to study. Is there any precedent for a snake phospholipase<br>
> > forming membrane channels? It would make sense physiologically,<br>
> > as a quick way to disrupt the membrane and place the phospholipase<br>
> > in the right environment to hydrolyze the lipids. One way that<br>
> > you could quickly look into this would be to add the phospholipase<br>
> > to artificial bilayers - one could maybe use ether-linked lipids<br>
> > to prevent hydrolysis and see if any pores are formed just by the<br>
> > presence of the toxin alone. We have a planar bilayer system in my lab,<br>
> > and I would be happy to give this a try if it would be of interest to<br>
> > you. As you mentioned, testing oligomerization in the presence of other<br>
> > detergents may also be interesting, including maybe lysolipids. Maybe<br>
> > we can set up a zoom call to discuss some of these questions in more<br>
> > detail if you are interested? Best, Sebastien"<br>
> ><br>
> > I am passing his questions on to this group to get your comments.<br>
> > Sebastien is now included on our email list, so please feel free to<br>
> > comment by simply replying to this message. I personally would be very<br>
> > curious to see if such a membrane pore theory could be tested with<br>
> > Sebastien's methods.<br>
> ><br>
> > I would like to follow Sebastien's suggestion and propose a zoom call to<br>
> > discuss next steps. Here is a Doodle poll for a zoom meeting, please<br>
> > respond by the end of tomorrow.<br>
> ><br>
> > <a href="https://doodle.com/poll/xbx5rhhf9g7mkxb3?utm_source=poll&utm_medium=link">
https://doodle.com/poll/xbx5rhhf9g7mkxb3?utm_source=poll&utm_medium=link</a><br>
> ><br>
> > If none of the proposed times work for most of us, we could try again<br>
> > next week with another doodle poll.<br>
> ><br>
> > Thanks! -Borries<br>
> > _______________________________________________<br>
> > Myotox mailing list<br>
> > Myotox@biophysics.uleth.ca<br>
> > <a href="https://demeler7.uleth.ca/mailman/listinfo/myotox">https://demeler7.uleth.ca/mailman/listinfo/myotox</a><br>
><br>
> --<br>
> Bruno Lomonte, Ph.D.<br>
> Instituto Clodomiro Picado<br>
> Facultad de Microbiología<br>
> Universidad de Costa Rica<br>
> San José, COSTA RICA<br>
><br>
> tel.of. (+506) 2511 7888<br>
> bruno.lomonte@ucr.ac.cr<br>
<br>
<br>
> _______________________________________________<br>
> Myotox mailing list<br>
> Myotox@biophysics.uleth.ca<br>
> <a href="https://demeler7.uleth.ca/mailman/listinfo/myotox">https://demeler7.uleth.ca/mailman/listinfo/myotox</a><br>
<br>
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