[Myotox] myotoxin2-SDS titration

[Borries Demeler]

As promised, here is an update on the SDS titration experiment.
Amy found plenty of Myotoxin for doing these experiments in our
freezer and performed an AUC experiment doing a titration with very low
concentration of SDS (0.01 and 0.02 percent of SDS, which should be well
below the CMC of SDS).  This clearly shows that there is a significant
change in the sedimentation profile (see attached).

A few notes:
1. The monomer control was actually run at 300 nm with ~21 mg/ml
concentration - so very high, and no evidence of dimerization there.
The protein concentration in the SDS titration experiments was much,
much lower at only about 10 uM.

2. even the 0.01% SDS sample exhibits significant shift in the s-values
which can only be explained by dimerization (as seen in the SDS-PAGE),
not by micelle formation with multiple myotoxin molecules being
incorporated into the micelle.

3. The 0.02% SDS concentration sample sedimented *slower* than the
0.01% SDS sample. If I think about this it makes sense if we
assume that instead of additional oligomerization we simply bind
more SDS to the complex, because SDS could significantly affect
friction. It also affects the partial specific volume. It is less
dense than protein so the overall partial specific volume of the
complex becomes less dense as more SDS is bound.

4. There isn't just one peak visible, consistent with either monomer or
dimer, but there are at least 2 major peaks in both samples, suggesting
possible even high order structures than dimer are present, or at least
some equilibrium between at least two oligomeric structures. It is not
clear to me what oligomers are present, but with a little additional
AUC work we could probably figure this out.

I think this brings up more questions, but also answers one important
question for Paul and Tony:
We can definitely significantly reduce the SDS concentration to avoid
the strong overlay signals from SDS in the NMR experiments. Also, we
can keep the very low phosphate concentration (5mM).

I think before we go to the next NMR experiments I want to do one
more AUC experiment: Amy, can you please find out the lowest possible
SDS concentration that causes a shift for a given protein concentration?

What I found surprising is that the 0.02% solution sedimented slower
than the 0.01%, this suggests the formation of discrete species, with
tight SDS binding, and no change in oligomerization between the two SDS
concentrations. That's important. I would like to know if we can find
out the lowest SDS concentration that causes this behavior.  I think
the SDS binds very tightly, so do we see the same behavior at 0.001%
SDS? I think we should do a titration one order of magnitude lower,
and at the same time increase the protein concentration. THis would
tell us two things: 1. what is the best ratio of high concentration
protein and low concentration SDS that clearly starts showing this
oligomerization? That ratio would be best for NMR, maximum protein signal,
minimum SDS signal. Let's find the critical ratios with AUC. We may have
to run the SDS titration with more points and also repeat it at
multiple protein concentrations. I would also like to know the maximum
s-value shift that can be observed, and whether the 2-peak pattern
persists.

Paul/Tony: What protein concentration would you like to run ideally?
1 mg/ml, 10 mg/ml, 20 mg/ml? Since Bruno is willing to send more sample,
I think we can play with this ratio a bit. Amy, how much is left?
For AUC I would prefer not to go much above 20 mg/ml to avoid
concentration dependent non-ideality effects.

Let me know what y'all think about this approach.

-Borries
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