[Myotox] myotoxin2-SDS titration

[Bruno Lomonte]

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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-- 
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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