[Myotox] SDS AUC update

Fri Mar 13 20:37:19 MDT 2020

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 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
>>
>
>> _______________________________________________
>> Myotox mailing list
>> Myotox at biophysics.uleth.ca
>> http://demeler7.uleth.ca/mailman/listinfo/myotox
>
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> 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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