[Myotox] [anagextra at gmail.com: Fwd: First draft - monomeric Mt-II]
Borries Demeler
demeler at gmail.com
Fri Dec 27 09:13:11 MST 2019
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
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