<div dir="ltr"><div dir="ltr">Sounds great ! 😀</div><div>Happy new year to everyone !</div><div>Warm regards,</div><div>Ana</div><br><div class="gmail_quote"><div dir="ltr" class="gmail_attr">Em sex., 27 de dez. de 2019 às 13:18, Borries Demeler <<a href="mailto:demeler@gmail.com">demeler@gmail.com</a>> escreveu:<br></div><blockquote class="gmail_quote" style="margin:0px 0px 0px 0.8ex;border-left:1px solid rgb(204,204,204);padding-left:1ex">Dear Ana,<br>
thanks for the detailed explanation, and the new draft.<br>
I took the liberty to subscribe others copied on this e-mail to our <br>
Myotoxin-II discussion list since this is very valuable information<br>
informing our planned experiments (you all should have received a <br>
notification of being subscribed to "<a href="mailto:myotox@biophysics.uleth.ca" target="_blank">myotox@biophysics.uleth.ca</a>",<br>
which everyone should use to correspond on this topic. You can find <br>
more information on this mailing list and manage your own subscription<br>
at: <a href="https://www.biophysics.uleth.ca/mailman/listinfo/myotox" rel="noreferrer" target="_blank">https://www.biophysics.uleth.ca/mailman/listinfo/myotox</a>)<br>
<br>
In light of the information from Ana I would strongly suggest to start<br>
with AUC experiments in the presence of lipids or SDS to see if we can<br>
simulate this induced dimerization by adding different lipids in small<br>
amounts (probably below CMC for easier interpretation). AUC will tell us<br>
how heterogeneous the mixtures are and if this is sufficient purity to<br>
expect useful data from a SAXS experiment.<br>
<br>
In any case, it will be straightforward and probably require less<br>
material to test if we test myotoxin-II by AUC in the presence of the<br>
lipids you mentioned.<br>
<br>
Perhaps as a first try we could measure the remaining amount of myotoxin<br>
at an intermediate concentration by titrating SDS into the mixture<br>
and see if this induces dimerization. If this works, we could also try<br>
some of the other lipids Anna mentioned. If there is not enough<br>
material, perhaps Bruno's lab could purify some more and send it to us.<br>
Amy will be back after the new year and can check into the remaining<br>
amounts. It is possible this was already passed on to Tony's lab for NMR<br>
experiments.<br>
<br>
How does that sound?<br>
<br>
-Borries<br>
<br>
<br>
Date: Thu, 26 Dec 2019 15:44:59 -0300<br>
From: Ana Gisele da Costa Neves Ferreira <<a href="mailto:anagextra@gmail.com" target="_blank">anagextra@gmail.com</a>><br>
To: Borries Demeler <<a href="mailto:demeler@gmail.com" target="_blank">demeler@gmail.com</a>>, Bruno Lomonte<br>
<<a href="mailto:bruno.lomonte@ucr.ac.cr" target="_blank">bruno.lomonte@ucr.ac.cr</a>>, Chico - UFRJ <<a href="mailto:gomes.netof@gmail.com" target="_blank">gomes.netof@gmail.com</a>>,<br>
"Henrickson, Amy" <<a href="mailto:amy.henrickson@uleth.ca" target="_blank">amy.henrickson@uleth.ca</a>>, Julian Fernandez Ulate<br>
<<a href="mailto:jfu273b@gmail.com" target="_blank">jfu273b@gmail.com</a>>, Emre Brookes <<a href="mailto:emre.brookes@umontana.edu" target="_blank">emre.brookes@umontana.edu</a>>,<br>
Richard <<a href="mailto:richardhemmi@gmail.com" target="_blank">richardhemmi@gmail.com</a>>, Thomas Jorgensen<br>
<<a href="mailto:tjdj@bmb.sdu.dk" target="_blank">tjdj@bmb.sdu.dk</a>>, Tatiana Brasil <<a href="mailto:tatiana.brasil@gmail.com" target="_blank">tatiana.brasil@gmail.com</a>>, Ana<br>
Gisele gmail <<a href="mailto:anagextra@gmail.com" target="_blank">anagextra@gmail.com</a>><br>
Subject: Fwd: First draft - monomeric Mt-II<br>
<br>
Dear co-authors,<br>
<br>
My comments/suggestions follow in the attached v03_AnaG draft. Since we<br>
have recently decided to publish the AUC data on the myotoxin separated<br>
from the data on the interaction with DM64, I´ve also included the names of<br>
Tatiana (SAXS analysis) and Richard/Thomas (MS analysis) as co-authors of<br>
this first paper (I have CC´d them on this email as well).<br>
<br>
As for molecular dynamics experiments (Tati, Richard and Thomas, please<br>
read the emails below to catch up with the story), Chico will need<br>
additional 2-3 weeks to have a better idea of the behavior of the "dimeric"<br>
myotoxin, he has just started to define the settings for the MD runs.<br>
<br>
Borries, regarding SAXS experiments on myotoxin in the presence of fatty<br>
acids, the literature on PLA2-like proteins (mainly based on crystal<br>
studies) assumes that dimerization of the toxin is key to toxicity. The<br>
hypothesis (still controversial) is that the toxins could exist as monomers<br>
in solution or as inactive dimers. The binding of fatty acids at their<br>
hydrophobic channel would induce monomer dimerization and/or allosteric<br>
changes from inactive ("conventional/unbound") to active<br>
("alternative/bound") dimers. The conformational change would occur through<br>
a molecular hinge, leading the bound toxins to expose/align a cationic<br>
"Membrane-Docking Site" (MDoS) and a hydrophobic "Membrane-Disruption Site<br>
(MDiS), which disrupts the lipid bilayer. Several PLA2-like structures have<br>
been solved in the absence/presence of saturated fatty acids [ex.: lauric<br>
acid (C12), myristic acid (C14), stearic acid (C18)], allowing for the<br>
study of these different oligomeric conformations (see attached review by<br>
Bruno and co-authors 2014). I have also attached a more recent paper in<br>
which the authors have used SDS (as a fatty acid mimetic) to trigger<br>
myotoxin oligomerization. Interestingly, they have monitored the<br>
oligomerization by AUC (Matsui 2019). In summary, either by SAXS and/or<br>
AUC, I think it would be nice to test for oligomerization in the presence<br>
of fatty acids/SDS. In any case, the AUC and SAXS data we already have<br>
clearly contradict the belief that myotoxin II exists as a "highly stable<br>
dimer in solution"; the papers that support this hypothesis were mainly<br>
based on electrophoresis and X-ray crystallography experiments. If I´m not<br>
wrong (Bruno ?), they were all run in the absence of fatty acids, just like<br>
we have done.<br>
<br>
Best regards and happy new year !<br>
Ana<br>
<br>
---------- Forwarded message ---------<br>
De: Ana Gisele da Costa Neves Ferreira <<a href="mailto:anagextra@gmail.com" target="_blank">anagextra@gmail.com</a>><br>
Date: dom., 22 de dez. de 2019 às 12:25<br>
Subject: Re: first draft - monomeric Mt-II<br>
To: Bruno Lomonte <<a href="mailto:bruno.lomonte@ucr.ac.cr" target="_blank">bruno.lomonte@ucr.ac.cr</a>><br>
Cc: Borries Demeler <<a href="mailto:demeler@gmail.com" target="_blank">demeler@gmail.com</a>>, Chico - UFRJ <<a href="mailto:gomes.netof@gmail.com" target="_blank">gomes.netof@gmail.com</a>>,<br>
Amy Henrickson <<a href="mailto:amy.henrickson@uleth.ca" target="_blank">amy.henrickson@uleth.ca</a>>, Julian Fernandez Ulate <<br>
<a href="mailto:jfu273b@gmail.com" target="_blank">jfu273b@gmail.com</a>>, Emre Brookes <<a href="mailto:emre.brookes@umontana.edu" target="_blank">emre.brookes@umontana.edu</a>><br>
<br>
<br>
Dear colleagues,<br>
I apologize for not having had time to touch the manuscript last week, I´m<br>
doing this today/tomorrow and will send my suggestions/comments as soon as<br>
possible.<br>
Best regards,<br>
AnaG<br>
<br>
Em sáb., 14 de dez. de 2019 às 02:02, Bruno Lomonte <<a href="mailto:bruno.lomonte@ucr.ac.cr" target="_blank">bruno.lomonte@ucr.ac.cr</a>><br>
escreveu:<br>
<br>
> Hi all,<br>
><br>
> here is the v.03 draft, reorganized after Borries' additions, so we can<br>
> use this new (provisional) version to add the data from Ana G and Chico<br>
><br>
> best regards to all,<br>
><br>
> Bruno<br>
><br>
><br>
><br>
> +++<br>
><br>
><br>
><br>
> On 13/12/2019 11:15, Borries Demeler wrote:<br>
> > Bruno, that sounds good. Let me see when we can get beamline access.<br>
> > I may have you send the SAXS samples to the beamline directly.<br>
> ><br>
> > Emre: would you have some beamline access available to get a myotoxin<br>
> > sample in phosphate buffer analyzed? If not, we could also do it with<br>
> > Trushar at the Saskatoon beamline.<br>
> ><br>
> > Ana: I am not clear on the purpose of running SAXS experiments on<br>
> myotoxin<br>
> > in the presence of fatty acids, can you explain the reasoning behind<br>
> this?<br>
> ><br>
> > Thanks, -Borries<br>
> ><br>
> ><br>
> > On Thu, Dec 12, 2019 at 10:38:52PM -0600, Bruno Lomonte wrote:<br>
> >> Great additions Borries! let me adjust the formatting (BBRC style)<br>
> >> and generate the next file (v.03) to pass it over to Ana G and Chico<br>
> >><br>
> >> as for the toxin, we better prepare a new batch to send it to you in<br>
> >> January, to use it in both SAXS and the NMR explorations - Julian<br>
> >> and I will work on it after the holidays break, and coordinate with<br>
> >> Amy like before<br>
> >><br>
> >> looking forward also to the MD simulations by Chico! great...<br>
> >><br>
> >> all the best,<br>
> >><br>
> >> Bruno<br>
> >><br>
> >><br>
> >> ++++<br>
> >><br>
> >><br>
> >><br>
> >> On 12/12/2019 22:06, Borries Demeler wrote:<br>
> >>> Dear all,<br>
> >>> Attached is a second version that includes the AUC analysis results and<br>
> >>> figures, and new endnotes/references.<br>
> >>><br>
> >>> I changed the author order around a bit, putting Amy first who did the<br>
> >>> majority of this AUC work.<br>
> >>><br>
> >>> Ana, I don't think we have enough material left to do SAXS analysis,<br>
> >>> plus we were going to use what we do have for preliminary NMR<br>
> >>> experiments. The MD results would be very useful, especially if they<br>
> >>> show monomer :)<br>
> >>><br>
> >>> Best wishes, -Borries<br>
> >>><br>
> >>> On Thu, Dec 12, 2019 at 04:54:12PM -0300, Ana Gisele da Costa Neves<br>
> Ferreira wrote:<br>
> >>>> Dear Bruno,<br>
> >>>> Thank you for the first draft, I´ve already taken a quick read. I´ll<br>
> >>>> complete the text parts regarding AUC and SAXS results in the next<br>
> few days<br>
> >>>> (they will be deleted from the DM64 manuscript). I talked to Chico<br>
> (cc)<br>
> >>>> earlier today and he came with two nice suggestions to enrich the<br>
> paper:<br>
> >>>> 1) He will try to run a molecular dynamics simulation on the dimeric<br>
> >>>> structure of myotoxin-II to investigate its behavior over time;<br>
> >>>> 2) Borries, if you have enough myotoxin-II left, do you think it<br>
> would be<br>
> >>>> possible to ask your colleagues in the US to repeat the SAXS analysis<br>
> in<br>
> >>>> the presence/absence of fatty acids ? If possible, we would check<br>
> which<br>
> >>>> fatty acids/conditions to assay.<br>
> >>>> Best regards,<br>
> >>>> Ana<br>
> >>>><br>
> >>>> Em qua., 11 de dez. de 2019 às 01:57, Bruno Lomonte <<br>
> <a href="mailto:bruno.lomonte@ucr.ac.cr" target="_blank">bruno.lomonte@ucr.ac.cr</a>><br>
> >>>> escreveu:<br>
> >>>><br>
> >>>>> Dear Ana Gisele and Borries,<br>
> >>>>><br>
> >>>>> I have tried to put together some ideas, in case we would like to<br>
> >>>>> present the evidence for a momomeric state in solution for the<br>
> Mt-II, in<br>
> >>>>> a short note<br>
> >>>>><br>
> >>>>> I have evaluated the possibilities of getting further proteins of<br>
> this<br>
> >>>>> type in sufficient quantities, to expland our findings, but concluded<br>
> >>>>> that it was not feasible for the moment<br>
> >>>>><br>
> >>>>> So please give it a thought if you think it is worth to try to<br>
> >>>>> communicate this first, before the larger study on the DM64-MtII<br>
> >>>>> interaction. I believe this manuscript could go to Biochem Biophys<br>
> Res<br>
> >>>>> Comm, but if you have any other thoughts they will be welcome<br>
> >>>>><br>
> >>>>> I anticipate that evaluation may not be so easy, since the findings<br>
> go<br>
> >>>>> against the widely accepted idea on the dimer state, and might imply<br>
> a<br>
> >>>>> substantial change in the view on how these toxins function - many<br>
> >>>>> years, many articles... experts may not be too happy :) but this<br>
> is<br>
> >>>>> science anyway...<br>
> >>>>><br>
> >>>>> You both would need to prepare the methods section and the results<br>
> >>>>> section, and integrate with the general discussion<br>
> >>>>><br>
> >>>>> Please consider that this is just a very first draft, a starting<br>
> point -<br>
> >>>>> modify at will !!! especially my poor English!!!<br>
> >>>>><br>
> >>>>> Let me hear your thoughts,<br>
> >>>>><br>
> >>>>> with best wishes for the holydays,<br>
> >>>>><br>
> >>>>> Bruno<br>
> >>>>><br>
> >>>>><br>
> >>>>><br>
> >>>>><br>
> >>>>><br>
> >>>>> --<br>
> >>>>> Bruno Lomonte, Ph.D.<br>
> >>>>> Instituto Clodomiro Picado<br>
> >>>>> Universidad de Costa Rica<br>
> >>>>> San José, 11501<br>
> >>>>> COSTA RICA<br>
> >>>>><br>
> >>>>> <a href="mailto:bruno.lomonte@ucr.ac.cr" target="_blank">bruno.lomonte@ucr.ac.cr</a><br>
> >>>>> tel. +506 2511 7888<br>
> >>>>> cel. +506 8392 0012<br>
> >>>>><br>
> >>>>><br>
> >>>> --<br>
> >>>> Ana Gisele da C. Neves Ferreira<br>
> >>>> Pesquisadora Titular em Saúde Pública<br>
> >>>> Chefe do Laboratorio de Toxinologia<br>
> >>>> Instituto Oswaldo Cruz - Fiocruz<br>
> >>>> Pavilhao Ozorio de Almeida, sala 05<br>
> >>>> Av. Brasil, 4365 - Manguinhos<br>
> >>>> Rio de Janeiro - RJ<br>
> >>>> 21040-900 Brasil<br>
> >>>> (+5521) 2562-1381 / 98801-5726<br>
> >> --<br>
> >> Bruno Lomonte, Ph.D.<br>
> >> Instituto Clodomiro Picado<br>
> >> Universidad de Costa Rica<br>
> >> San José, 11501<br>
> >> COSTA RICA<br>
> >><br>
> >> <a href="mailto:bruno.lomonte@ucr.ac.cr" target="_blank">bruno.lomonte@ucr.ac.cr</a><br>
> >> tel. +506 2511 7888<br>
> >> cel. +506 8392 0012<br>
> >><br>
> --<br>
> Bruno Lomonte, Ph.D.<br>
> Instituto Clodomiro Picado<br>
> Universidad de Costa Rica<br>
> San José, 11501<br>
> COSTA RICA<br>
><br>
> <a href="mailto:bruno.lomonte@ucr.ac.cr" target="_blank">bruno.lomonte@ucr.ac.cr</a><br>
> tel. +506 2511 7888<br>
> cel. +506 8392 0012<br>
><br>
><br>
<br>
-- <br>
Ana Gisele da C. Neves Ferreira<br>
Pesquisadora Titular em Saúde Pública<br>
Chefe do Laboratorio de Toxinologia<br>
Instituto Oswaldo Cruz - Fiocruz<br>
Pavilhao Ozorio de Almeida, sala 05<br>
Av. Brasil, 4365 - Manguinhos<br>
Rio de Janeiro - RJ<br>
21040-900 Brasil<br>
(+5521) 2562-1381 / 98801-5726<br>
<br>
<br>
<br>
<br>
<br>
-- <br>
Ana Gisele da C. Neves Ferreira<br>
Pesquisadora Titular em Saúde Pública<br>
Chefe do Laboratorio de Toxinologia<br>
Instituto Oswaldo Cruz - Fiocruz<br>
Pavilhao Ozorio de Almeida, sala 05<br>
Av. Brasil, 4365 - Manguinhos<br>
Rio de Janeiro - RJ<br>
21040-900 Brasil<br>
(+5521) 2562-1381 / 98801-5726<br>
<br>
<br>
<br>
<br>
<br>
-- <br>
Ana Gisele da C. Neves Ferreira<br>
Pesquisadora Titular em Saúde Pública<br>
Chefe do Laboratorio de Toxinologia<br>
Instituto Oswaldo Cruz - Fiocruz<br>
Pavilhao Ozorio de Almeida, sala 05<br>
Av. Brasil, 4365 - Manguinhos<br>
Rio de Janeiro - RJ<br>
21040-900 Brasil<br>
(+5521) 2562-1381 / 98801-5726<br>
<br>
<br>
<br>
<br>
<br>
-- <br>
Ana Gisele da C. Neves Ferreira<br>
Pesquisadora Titular em Saúde Pública<br>
Chefe do Laboratorio de Toxinologia<br>
Instituto Oswaldo Cruz - Fiocruz<br>
Pavilhao Ozorio de Almeida, sala 05<br>
Av. Brasil, 4365 - Manguinhos<br>
Rio de Janeiro - RJ<br>
21040-900 Brasil<br>
(+5521) 2562-1381 / 98801-5726<br>
<br>
<br>
<br>
<br>
<br>
-- <br>
Ana Gisele da C. Neves Ferreira<br>
Pesquisadora Titular em Saúde Pública<br>
Chefe do Laboratorio de Toxinologia<br>
Instituto Oswaldo Cruz - Fiocruz<br>
Pavilhao Ozorio de Almeida, sala 05<br>
Av. Brasil, 4365 - Manguinhos<br>
Rio de Janeiro - RJ<br>
21040-900 Brasil<br>
(+5521) 2562-1381 / 98801-5726<br>
<br>
<br>
<br>
<br>
<br>
<br>
<br>
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</blockquote></div><br clear="all"><div><br></div>-- <br><div dir="ltr" class="gmail_signature"><div dir="ltr"><div><div dir="ltr"><div dir="ltr"><div dir="ltr"><div><span style="color:rgb(11,83,148);font-family:tahoma,sans-serif;font-size:x-small">Ana Gisele da C. Neves Ferreira</span><br></div><div><font style="background-color:rgb(255,255,255)" face="tahoma, sans-serif" color="#0b5394" size="1">Pesquisadora Titular em Saúde Pública</font></div><div><font style="background-color:rgb(255,255,255)" face="tahoma, sans-serif" color="#0b5394" size="1">Chefe do Laboratorio de Toxinologia</font></div><div><font style="background-color:rgb(255,255,255)" face="tahoma, sans-serif" color="#0b5394" size="1">Instituto Oswaldo Cruz - Fiocruz</font></div><div><font style="background-color:rgb(255,255,255)" face="tahoma, sans-serif" color="#0b5394" size="1">Pavilhao Ozorio de Almeida, sala 05</font></div><div><font style="background-color:rgb(255,255,255)" face="tahoma, sans-serif" color="#0b5394" size="1">Av. Brasil, 4365 - Manguinhos</font></div><div><font style="background-color:rgb(255,255,255)" face="tahoma, sans-serif" color="#0b5394" size="1">Rio de Janeiro - RJ </font></div><div><font style="background-color:rgb(255,255,255)" face="tahoma, sans-serif" color="#0b5394" size="1">21040-900 Brasil</font></div><div><font style="background-color:rgb(255,255,255)" face="tahoma, sans-serif" color="#0b5394" size="1">(+5521) 2562-1381 / 98801-5726</font></div><div style="color:rgb(136,136,136);font-size:12.8px"><font size="1" color="#666666" face="tahoma, sans-serif"><br><br><br></font></div></div></div></div></div></div></div></div>