[Myotox] myotoxin2-SDS titration

Sun Mar 21 16:37:32 MDT 2021

Hi Borries
For standard NMR tubes we typically make up 600-650 uL of the solution.
Then we put 550uL in the NMR tube
But this work is being done in a Shegimi tube, which uses far less volume, as it restricts the sample to the active coil area only. This increases the sensitivity and the glass in the shegimi tube is also susceptibility matched for d2o/h2o which allows for better shimming.
For these tubes we need around 280-320uL of sample. Maybe plan for 350uL just to be safe.
Mike, can you please confirm if I am correct on the shegimi tube volumes ?
Cheers
Tony

Tony Montina
Director, Science Operations
Director, Magnetic Resonance Facility
Faculty of Arts and Science
Instructor, Department of Chemistry and Biochemistry
University of Lethbridge
SA6214, Science Commons
4401 University Drive West
Lethbridge, Alberta, Canada, T1K3M4
Office: 1-403-394-3927
Lab: 1-403-329-2230

This message has been sent from my mobile device so please ignore any autocorrect or typing errors.

On Mar 21, 2021, at 4:08 PM, Borries Demeler <demeler at gmail.com> wrote:

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Thank you, Tony!
Can you remind me of the volume needed to do the experiment you planned?
This will determine if we need Bruno to send more protein.

Thanks, -Borries

On Sun, Mar 21, 2021 at 06:43:18PM +0000, Montina, Tony wrote:
Hello All,
I agree with both Paul and Borries.
The higher we can get the myotoxin concentration in SDS the better for signal to noise and overall NMR experiment time in our room temperature probe. Also, as Paul and Borries both mentioned, using a 5mM concentration of the buffer and also a significantly lower SDS concentration (at least one order of magnitude but potentially two) will greatly improve the dynamic range of the ADC convertor and hence should result in better signal to noise (and possibly resolution).
The last experiments we used ~2.5mg/mL concentration and our first experiments used 4 mg/ml.
This was because things started precipitating out at any higher of concentration. Hopefully this was due to the micelle formation and once this problem is gone - now that we are working with SDS below the CMC - we can get myotoxin into solution at much higher concentrations.
First, Borries and Amy can determine the minimum amount of SDS required that is below the 0.2% CMC of SDS.
I would then suggest that Amy and Mike use this SDS concentration to determine the max solubility of the myotoxin in that SDS concentration and 5mM buffer (but not exceed 20mg/mL of myotoxin as suggested by Borries).
Once this is done, we can re-run the NMR experiments on the SDS (at lower concentration) + Myotoxin (hopefully at a higher concentration) in 5mM buffer. Hopefully we can get the concentration of myotoxin up much higher than 4mg/mL so that we can run these experiments quickly.
Amy, do we have enough myotoxin on hand in Lethbridge to go up to 20mg/mL?
Lastly, I agree that a NOESY experiment will be useful, but that would be more useful for the later paper and depending on the final concentration of myotoxin we are able to use in SDS, may or may not be possible.
For example, if this is a 2 week long NOESY experiment we may not be able to run this on our room temperature probe. Increasing the concentration will help greatly with this problem.
I hope that helps clear up the sample prep questions for NMR.
Amy, please reach out to Mike and I when you are ready next week and we can get the new sample prepared.
This is really good timing, as I believe Mike has booked the NMR instrument for the entirety of next weekend.
Cheers
Tony
________________________________________
Tony Montina
Director | Science Operations
Director | Magnetic Resonance Facility
Instructor | Department of Chemistry and Biochemistry
Faculty of Arts and Science
The University of Lethbridge
Lethbridge, Alberta, Canada, T1K 3M4
Office: 1-403-394-3927
Lab: 1-403-329-2230

-----Original Message-----
From: Myotox <myotox-bounces at biophysics.uleth.ca> On Behalf Of Hazendonk, Paul
Sent: March 20, 2021 10:41 PM
To: Myotoxin-II discussion <myotox at biophysics.uleth.ca>
Subject: Re: [Myotox] myotoxin2-SDS titration

As high a conc of protein as is possible, considering aggregation issues etc, etc.  It will really speed things up especially the 2D experiments.

-----Original Message-----
From: Myotox <myotox-bounces at biophysics.uleth.ca> On Behalf Of Borries Demeler
Sent: March 20, 2021 9:38 PM
To: Myotoxin-II discussion <myotox at biophysics.uleth.ca>
Subject: Re: [Myotox] myotoxin2-SDS titration

Caution: This email was sent from someone outside of the University of Lethbridge. Do not click on links or open attachments unless you know they are safe. Suspicious emails should be forwarded to phishing at uleth.ca.

If this is possible that would be very exciting! Paul, can you please indicate the concentration and volume of protein needed to perform these experiments. We will then test by AUC how low we can go with SDS and still see the shift in s.

-Borries
On Sun, Mar 21, 2021 at 02:36:30AM +0000, Paul Hazendonk wrote:
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

Caution: This email was sent from someone outside of the University of Lethbridge. Do not click on links or open attachments unless you know they are safe. Suspicious emails should be forwarded to phishing at uleth.ca<mailto:phishing at uleth.ca>.

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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