[Myotox] myotoxin2-SDS titration

[Borries Demeler]

Bruno, thanks very much. Before you ship more protein, let's hear how
much Amy still has and how much we'll need for the follow-up NMR
experiments. Amy/Paul/Tony, can you please check?
Amy for the next titration please include a simple monomer control 
to make sure the protein still is in good shape.

Thanks, and a pleasant remainder of the weekend to everyone! -Borries

On Sat, Mar 20, 2021 at 04:39:45PM -0600, Bruno Lomonte wrote:
> Interesting as always, Borries and all !  the combination of your
> biophysical analyses + NMR are providing lots of exciting new information,
> as you explained. I will send more protein this week to Amy's address.
> Thanks a lot for all the interest and hard work.
> 
> Best regards,
> 
> Bruno
> 
> 
> +++++++
> 
> On 3/20/2021 11:20 AM, Borries Demeler wrote:
> > 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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> > Myotox at biophysics.uleth.ca
> > https://demeler7.uleth.ca/mailman/listinfo/myotox
> 
> -- 
> Bruno Lomonte, Ph.D.
> Instituto Clodomiro Picado
> Facultad de Microbiología
> Universidad de Costa Rica
> San José, COSTA RICA
> 
> tel.of. (+506) 2511 7888
> bruno.lomonte at ucr.ac.cr
> 

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