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Thanks Borries, and yes, we can try to explore the gel behavior at
various SDS concentrations with the help of a student here.<br>
<br>
By the way, Prof. Marcos Fontes (whom you have met in the recent
Zoom calls) is here in Costa Rica to deliver a mini-course on
protein structure for our students, and we are having good times in
updating ideas on the snake "Lys49" myotoxins!<br>
<br>
Best regards to all,<br>
<br>
Bruno<br>
<br>
++++++++++++++++++++++++++++++++<br>
<br>
<br>
<br>
<div class="moz-cite-prefix">On 8/22/2022 12:26 PM, Borries Demeler
wrote:<br>
</div>
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<div>Hi Bruno,</div>
<div>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.<br>
</div>
<div><br>
</div>
<div>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?</div>
<div>-Borries<br>
</div>
<div><br>
</div>
<div><br>
</div>
<div><br>
</div>
</div>
<br>
<div class="gmail_quote">
<div dir="ltr" class="gmail_attr">On Sun, Aug 21, 2022 at
10:19 PM Bruno Lomonte <<a
href="mailto:bruno.lomonte@ucr.ac.cr"
moz-do-not-send="true" class="moz-txt-link-freetext">bruno.lomonte@ucr.ac.cr</a>>
wrote:<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 Borries and all,<br>
<br>
the data and conclusions reached thus far seem quite
impressive to me, <br>
so I tend to believe this would be a good moment to put it
together in a <br>
report for EBJ as suggested<br>
<br>
I would only have two very naive questions to you experts on
these <br>
biophysical aspects:<br>
<br>
1 - would it be worth to see the behavior of Mt-II in a
series of <br>
SDS-PAGE gels having the same final SDS concentrations as
those tested <br>
in the AUC experiments? for example by casting a gel with
several <br>
spacers and run side-by-side the Mt-II varying only the SDS?
I am not <br>
sure of what will happen or how useful the results would be,
it is <br>
mostly a curiosity for visualizing what would happen as
predicted by the <br>
AUC studies<br>
<br>
2 - in the simulations by Francisco, would it be of interest
to also try <br>
the same in silico experiment using the coordinates of Mt-II
reported in <br>
the suramin complex (which was reported to have a different
dimeric <br>
organization), 1Y4L, I would guess eliminating the suramin
from the <br>
data, to see what is the outcome and compare with that of
1CLP? again, I <br>
am not sure if this could provide any useful information to
this part of <br>
the study, just a curiosity because it could be that the
1CLP crystal <br>
dimer perhaps can be interpreted alternatively in its
dimerization as a <br>
"compact" instead of the "extended" structure (as proposed
by Fontes' <br>
group) - would that influence the outcome?<br>
<br>
best regards to all, and thank you Borries for nicely
integrating all <br>
that has been done until now!<br>
<br>
Bruno<br>
<br>
+++++++++++++++++++++++<br>
<br>
<br>
On 8/21/2022 5:37 PM, Borries Demeler wrote:<br>
> Dear Colleagues,<br>
> 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:<br>
><br>
> 1. By AUC, MT-II remains monomeric in PBS up to very
high concentrations (I believe 20 mg/ml was the highest we
studied),<br>
> 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<br>
> 3. In AUC, we observed formation of a discrete hexamer
or heptamer of MT-II in the presence of very low SDS.<br>
> 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.<br>
> 5. The same effect could not be reproduced with other
lipids or detergents<br>
> 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.<br>
> 7. electrophysiology experiments with bilayer membranes
did not produce results - Sebastien, are there any updates?<br>
> 8. negative staining and cryoEM turned out to be a dead
end<br>
> 9. MD simulations involving SDS by Francisco suggest a
dimer formation<br>
><br>
> 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.<br>
><br>
> I'm open to any and all suggestions and would like to
get your feedback.<br>
><br>
> I'm also attaching a summary of what we have from
Francisco and from our lab.<br>
><br>
> Looking forward to get your feedback.<br>
><br>
> Regards, -Borries<br>
><br>
> _______________________________________________<br>
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<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>
<a href="mailto:bruno.lomonte@ucr.ac.cr" target="_blank"
moz-do-not-send="true" class="moz-txt-link-freetext">bruno.lomonte@ucr.ac.cr</a><br>
<br>
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<pre class="moz-signature" cols="72">--
Bruno Lomonte, Ph.D.
Instituto Clodomiro Picado
Facultad de Microbiología
Universidad de Costa Rica
San José, COSTA RICA
tel.of. (+506) 2511 7888
<a class="moz-txt-link-abbreviated" href="mailto:bruno.lomonte@ucr.ac.cr">bruno.lomonte@ucr.ac.cr</a></pre>
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