[Myotox] SDS AUC update

[Borries Demeler]

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