[Myotox] SDS AUC update

Sat May 9 10:27:29 MDT 2020

Hello everyone,

@Borries: the latest file I have for the draft manuscript is the 
attached v06 with Amy's notes

best regards to all and take care with the virus!

Bruno

+++

On 5/9/2020 9:48 AM, Borries Demeler wrote:
> On Fri, May 08, 2020 at 06:34:49PM -0300, Ana Gisele da Costa Neves Ferreira wrote:
>> Dear Borries and colleagues,
>> I hope this email finds you and your families well, safe from COVID-19.
>> Currently, our lab team in Brazil is almost exclusively working from home.
>> Thinking about pending manuscripts, I was wondering whether you have had
>> the chance to generate new AUC/NMR data on myotoxin II before the pandemic.
> Hi Ana,
> Yes, our last AUC data was the run Amy did with myotoxin in the presence
> of SDS which conclusively showed that myotoxin dimerizes in the presence
> of SDS, and that this was apparently reversible/mass action dependent,
> if I recall correctly - need to review the data. Amy also provided
> myotoxin to Tony and Paul, but I am not sure what the status is on this,
> can you guys fill us in?
>
>> Anyway, I think we already have a very interesting set of results and we
>> should think about submitting a first manuscript. We also have nice
>> structural data on the complex made of myotoxin II and the inhibitor DM64
>> that needs to be published ASAP (following the myotoxin paper).
>> Please let me know what you think.
> Bruno started the manuscript and we should all review where we are and
> get feedback from Paul to see if there is any NMR data that could be
> included, if not, I agree that it has been a long time in the making and
> we should proceed to publishing now. I think we have enough for a
> publication now, I would just like to know if it can be made strong by
> NMR data. So, Paul, can you please comment? Also, who has the latest
> version that we could all review?
>
> -Borries
>
>
>
>> Best regards,
>> AnaG
>>
>> Em sex., 13 de mar. de 2020 às 23:45, Hazendonk, Paul <
>> paul.hazendonk at uleth.ca> escreveu:
>>
>>> I think relaxation dispersion experiments should be able to shed some
>>> light on this.
>>>
>>> Get Outlook for Android <https://aka.ms/ghei36>
>>>
>>> ------------------------------
>>> *From:* Myotox <myotox-bounces at biophysics.uleth.ca> on behalf of Bruno
>>> Lomonte <bruno.lomonte at ucr.ac.cr>
>>> *Sent:* Friday, March 13, 2020 8:37:19 PM
>>> *To:* myotox at biophysics.uleth.ca <myotox at biophysics.uleth.ca>
>>> *Subject:* Re: [Myotox] SDS AUC update
>>>
>>>
>>> Fantastic that the paper helped Borries. And great that the results fitted
>>> well!
>>>
>>> As you know, I feel a bit overwhelmed by these biophysical calculations
>>> and techniques, because of my different background, so I have little to
>>> say. But at the same time I feel so impressed for all the information that
>>> these methods can provide! just great!
>>>
>>> Good luck in the next round of tests. If you succeed altogether to define
>>> the behaviour of this K49 protein regarding the monomer/dimer state
>>> 'dilemma' in solution, it will certainly be an important step forward and
>>> contribution to better understand its mechanism of action on cell membranes.
>>>
>>> Congratulations, and best regards to all
>>>
>>> Bruno
>>>
>>>
>>> +++
>>>
>>>
>>>
>>> On 3/13/2020 5:07 PM, Borries Demeler wrote:
>>>
>>> Bruno,
>>> this paper answered many of my questions, thank you very much for
>>> forwarding this. I would be curious if Paul's group could comment
>>> on what portions of these findings could be confirmed with NMR.
>>>
>>> Meanwhile, I have refined our AUC analysis of the SDS results further.
>>> Attached is a plot of a genetic algorithm/Monte Carlo analysis that
>>> shows the s-value distributions from the 4 experiments Amy ran. The
>>> high concentration protein + 1% SDS shows a peak that seems to suggest
>>> an oligomerization endpoint with s=3.1791e-13 s and D=8.0428e-07.
>>> Molar mass interpretations require knowledge of partial specific volume,
>>> which is discussed in the Uchiyama paper.
>>>
>>> Based on some of the estimates of vbar for SDS (0.87 ml/g) and the molar
>>> mass of an SDS monomer (~288 Da), it is now possible to calculate the
>>> ratio of SDS to myotoxin that results in a molar mass and vbar that is
>>> consistent with the hydrodynamic measurements. Only one ratio of protein
>>> and SDS will give a match. I wrote a small program to find that (using
>>> the vbar of SDS cited in the Uchiyama paper) and determined the protein
>>> MUST be dimeric and the number of SDS molecules must be 55 (tested
>>> between 0-70 bound SDS molecules). There is no other solution to this
>>> equation (see attached plots of monomer, dimer and trimer scenarios).
>>>
>>> The obtained difference in molar mass is 13 dalton at the match point.
>>> The attached images show the overlays of sum of molar masses, and
>>> the molar mass obtained from the Svedberg equation when we use the
>>> theoretical vbar from the combination of protein and SDS.  Furthermore,
>>> the 55 molecules of SDS agrees very well with Uchiyama's paper, and
>>> the vbar at this point is 0.779 ml/g, very close to the measured value
>>> reported by Uchiyama. These numbers are dependent on the assumption
>>> that the vbar of SDS is 0.87 ml/g
>>>
>>> Therefore, I conclude that this larger species must be a dimer, and not
>>> a monomer or trimer.
>>>
>>> Going forward, Amy will measure the vbar using D2O density matching
>>> experiments on the dimer at a slightly higher protein concentration
>>> to push everything solidly to the dimer, and also measure at very low
>>> concentration so we can see pure monomer in SDS and measure the number
>>> of SDS molecules bound at that point. Based on these data we may even
>>> be able to measure Kds for this association in the presence of SDS.
>>>
>>> I like it when data make good sense!
>>>
>>> If it were worthwhile, we could also do the fluorescence anisotropy
>>> measurements in Missoula, where we have access to a very good
>>> FL spectroscopy lab. Let me know if this would be of interest.
>>> At this point I would be most interested in the information we could
>>> learn from NMR. Paul, Tony??
>>>
>>> -Borries
>>>
>>>
>>> On Thu, Mar 12, 2020 at 08:29:27PM -0600, Bruno Lomonte wrote:
>>>
>>> I see Borries... agree
>>>
>>> I found this 2007 paper that perhaps gives us important clues on this
>>> phenomenon of SDS-toxin interaction?
>>>
>>> the authors put forward the idea that SDS 'simulates' the effect of
>>> phospholipid interactions on the protein, and that this would have relevance
>>> to the membrane-damaging mechanism
>>>
>>> please see attached
>>>
>>> Bruno
>>>
>>>
>>> ++++
>>>
>>>
>>>
>>> On 3/12/2020 8:12 PM, Borries Demeler wrote:
>>>
>>> On Thu, Mar 12, 2020 at 07:26:21PM -0600, Bruno Lomonte wrote:
>>>
>>> Dear colleagues
>>>
>>>
>>> thanks for running these new experiments, and finding such interesting
>>> results!
>>>
>>>
>>> so this dimerization induced by SDS would explain the appearance of a smear
>>> around the dimeric value in such electrophoretic analyses?   and this would
>>> support that under physiological conditions the toxin would be a monomer,
>>> the dimer being an artifact of crystallization perhaps? I am not sure if I
>>> am interpreting this correctly
>>>
>>> Dear Bruno,
>>> I don't think the interpretation is straightforward. SDS is having
>>> a strange effect on this protein, whose structural basis should be
>>> understood. Uchiyama's group found the same issue on their protein.
>>> Other than these two proteins I have never heard that SDS is causing
>>> proteins to dimerize. Furthermore, Myotoxin-II is highly soluble even
>>> at very high concentrations, it doesn't need SDS to be in solution. ALL
>>> proteins I know of *monomerize* when mixed with SDS, at least on SDS-PAGE
>>> gels. That's the exact opposite of what we are seeing here. SDS is a
>>> strong denaturant.
>>>
>>> I think the questions we need to answer is this:
>>> 1. why is the protein oligomerizing with an apparent end state (dimer or
>>> trimer?) when exposed to SDS? What is the structural basis for this?
>>>
>>> 2. is this behavior also observed when the protein is mixed with other
>>> lipids?
>>>
>>> 3. is this behavior somehow related to its toxic action, perhaps
>>> disrupting membranes?
>>>
>>> 4. What does the protein do when/if it binds to membranes?
>>>
>>> The Kd for oligomerization appears to be quite low. We will investigate
>>> this further by running in 1% (thanks, Amy, for the correction!) SDS
>>> again, but this time at lower concentration to see if it can push it to
>>> pure monomer in SDS. The process of oligomerization appears to be
>>> completely reversible and mass action driven.
>>>
>>>
>>> exciting news indeed
>>>
>>> Yes, fascinating. Susumu Uchiyama is a good friend of mine, I didn't
>>> even realize he was on the paper until today, he probably did the mass
>>> spec and AUC analysis, which, by the way, was well done. I will ask him
>>> if he has any further insights into the action of SDS.
>>>
>>> Regards, -Borries
>>> _______________________________________________
>>> Myotox mailing listMyotox at biophysics.uleth.cahttp://demeler7.uleth.ca/mailman/listinfo/myotox
>>>
>>> --
>>> 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
>>>
>>>
>>> _______________________________________________
>>> Myotox mailing listMyotox at biophysics.uleth.cahttp://demeler7.uleth.ca/mailman/listinfo/myotox
>>>
>>>
>>> _______________________________________________
>>> Myotox mailing listMyotox at biophysics.uleth.cahttp://demeler7.uleth.ca/mailman/listinfo/myotox
>>>
>>> --
>>> 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
>>>
>>>
>>> _______________________________________________
>>> Myotox mailing list
>>> 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
>> _______________________________________________
>> Myotox mailing list
>> Myotox at biophysics.uleth.ca
>> http://demeler7.uleth.ca/mailman/listinfo/myotox
> _______________________________________________
> Myotox mailing list
> Myotox at biophysics.uleth.ca
> http://demeler7.uleth.ca/mailman/listinfo/myotox

-- 
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

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