[Myotox] Manuscript?

Mon Aug 22 20:00:08 MDT 2022

Thanks Borries, and yes, we can try to explore the gel behavior at 
various SDS concentrations with the help of a student here.

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!

Best regards to all,

Bruno

++++++++++++++++++++++++++++++++

On 8/22/2022 12:26 PM, Borries Demeler wrote:
> Hi Bruno,
> 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.
>
> 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?
> -Borries
>
>
>
>
> On Sun, Aug 21, 2022 at 10:19 PM Bruno Lomonte 
> <bruno.lomonte at ucr.ac.cr> wrote:
>
>     Dear Borries and all,
>
>     the data and conclusions reached thus far seem quite impressive to
>     me,
>     so I tend to believe this would be a good moment to put it
>     together in a
>     report for EBJ as suggested
>
>     I would only have two very naive questions to you experts on these
>     biophysical aspects:
>
>     1 - would it be worth to see the behavior of Mt-II in a series of
>     SDS-PAGE gels having the same final SDS concentrations as those
>     tested
>     in the AUC experiments? for example by casting a gel with several
>     spacers and run side-by-side the Mt-II varying only the SDS? I am not
>     sure of what will happen or how useful the results would be, it is
>     mostly a curiosity for visualizing what would happen as predicted
>     by the
>     AUC studies
>
>     2 - in the simulations by Francisco, would it be of interest to
>     also try
>     the same in silico experiment using the coordinates of Mt-II
>     reported in
>     the suramin complex (which was reported to have a different dimeric
>     organization), 1Y4L, I would guess eliminating the suramin from the
>     data, to see what is the outcome and compare with that of 1CLP?
>     again, I
>     am not sure if this could provide any useful information to this
>     part of
>     the study, just a curiosity because it could be that the 1CLP crystal
>     dimer perhaps can be interpreted alternatively in its dimerization
>     as a
>     "compact" instead of the "extended" structure (as proposed by Fontes'
>     group) - would that influence the outcome?
>
>     best regards to all, and thank you Borries for nicely integrating all
>     that has been done until now!
>
>     Bruno
>
>     +++++++++++++++++++++++
>
>
>     On 8/21/2022 5:37 PM, Borries Demeler wrote:
>     > Dear Colleagues,
>     > 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:
>     >
>     > 1. By AUC, MT-II remains monomeric in PBS up to very high
>     concentrations (I believe 20 mg/ml was the highest we studied),
>     > 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
>     > 3. In AUC, we observed formation of a discrete hexamer or
>     heptamer of MT-II in the presence of very low SDS.
>     > 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.
>     > 5. The same effect could not be reproduced with other lipids or
>     detergents
>     > 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.
>     > 7. electrophysiology experiments with bilayer membranes did not
>     produce results - Sebastien, are there any updates?
>     > 8. negative staining and cryoEM turned out to be a dead end
>     > 9. MD simulations involving SDS by Francisco suggest a dimer
>     formation
>     >
>     > 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.
>     >
>     > I'm open to any and all suggestions and would like to get your
>     feedback.
>     >
>     > I'm also attaching a summary of what we have from Francisco and
>     from our lab.
>     >
>     > Looking forward to get your feedback.
>     >
>     > Regards, -Borries
>     >
>     > _______________________________________________
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>     > Myotox at biophysics.uleth.ca
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>
>     -- 
>     Bruno Lomonte, Ph.D.
>     Instituto Clodomiro Picado
>     Facultad de Microbiología
>     Universidad de Costa Rica
>     San José, COSTA RICA
>
>     tel.of. (+506) 2511 7888
>     bruno.lomonte at ucr.ac.cr
>
>
> _______________________________________________
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> Myotox at biophysics.uleth.ca
> https://demeler7.uleth.ca/mailman/listinfo/myotox

-- 
Bruno Lomonte, Ph.D.
Instituto Clodomiro Picado
Facultad de Microbiología
Universidad de Costa Rica
San José, COSTA RICA

tel.of. (+506) 2511 7888
bruno.lomonte at ucr.ac.cr
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