[Myotox] myotoxin2-SDS titration

Sat Mar 20 20:36:30 MDT 2021

This is very exciting.  May I suggest we also try a NOESY experiment one we have done the analysis on the low sds conc. Sample since if the binding is very specific we may be able to identify the amino acids with which it is interacting.

From: Myotox <myotox-bounces at biophysics.uleth.ca> On Behalf Of Bruno Lomonte
Sent: March 20, 2021 4:40 PM
To: myotox at biophysics.uleth.ca
Subject: Re: [Myotox] myotoxin2-SDS titration

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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<mailto:bruno.lomonte at ucr.ac.cr>
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