[Myotox] [anagextra at gmail.com: Fwd: First draft - monomeric Mt-II]

Fri Dec 27 11:44:05 MST 2019

Sounds great ! 😀
Happy new year to everyone !
Warm regards,
Ana

Em sex., 27 de dez. de 2019 às 13:18, Borries Demeler <demeler at gmail.com>
escreveu:

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