[Myotox] SDS AUC update

Fri Mar 13 20:45:18 MDT 2020

I think relaxation dispersion experiments should be able to shed some light on this.

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________________________________
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
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Bruno Lomonte, Ph.D.
Instituto Clodomiro Picado
Universidad de Costa Rica
San José, 11501
COSTA RICA

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Myotox at biophysics.uleth.ca<mailto:Myotox at biophysics.uleth.ca>
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--
Bruno Lomonte, Ph.D.
Instituto Clodomiro Picado
Universidad de Costa Rica
San José, 11501
COSTA RICA

bruno.lomonte at ucr.ac.cr<mailto:bruno.lomonte at ucr.ac.cr>
tel. +506  2511 7888
cel. +506  8392 0012

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