[Myotox] myotoxin2-SDS titration

Mon Mar 22 15:41:05 MDT 2021

Amy, can you please make sure Bruno has the necessary customs pre-clearance
info?
Thanks very much, Bruno!

Also, with the remaining material let's run the SDS titration. To minimize
protein requirement, perhaps run the tests at lower protein concentration.
I think we should run at 0.5 OD 280 nm (~25 uM) with 0.001, 0.002, 0.005,
0.01, 0.05, and 0.1 % SDS, 6 *samples, 3 cells*. Run at 50,000 rpm.
Thanks! -Borries

On Mon, Mar 22, 2021 at 2:22 PM Bruno Lomonte <bruno.lomonte at ucr.ac.cr>
wrote:

> Sure! 11.0 mg to be shipped by courier tomorrow...
>
> thanks to all
>
> Bruno
>
>
> +++
>
>
> On 22/03/2021 01:33 p. m., Montina, Tony wrote:
>
> Hello Amy,
>
> I guess we should use as much myotoxin as possible this time around for
> the NMR measurements.
>
> We can use this sample for the NMR time booked this weekend.
>
> Once more arrives, we should try to further concentrate the NMR sample to
> the 20mg/mL.
>
> Bruno, this works out to almost 10mg of myotoxin, plus what Amy needs for
> AUC.
>
> Are you able to send this large of a quantity?
>
> 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
>
>
>
> *From:* Myotox <myotox-bounces at biophysics.uleth.ca>
> <myotox-bounces at biophysics.uleth.ca> *On Behalf Of *Henrickson, Amy
> *Sent:* March 22, 2021 12:33 PM
> *To:* Myotoxin-II discussion <myotox at biophysics.uleth.ca>
> <myotox at biophysics.uleth.ca>
> *Subject:* Re: [Myotox] myotoxin2-SDS titration
>
>
>
> Hello All,
>
>
>
> There is only about 3-4mg total.  So we could possibly get ~6mg/ml by
> diluting into 500uL, but this would leave every little if any for the
> further AUC tests.
>
>
>
> Therefore, Bruno if you do have some that you could ship that would be
> greatly appreciated.
>
>
>
> The address that it can be shipped to is:
>
> Attn: Amy Henrickson
>
> 4401 University Drive W
>
> TIK 3M4, Lethbridge, AB
>
> Canada
>
>
>
> Thanks,
>
>
>
> Amy Henrickson
> ------------------------------
>
> *From:* Myotox <myotox-bounces at biophysics.uleth.ca> on behalf of Opyr,
> Michael <michael.opyr at uleth.ca>
> *Sent:* Monday, March 22, 2021 9:08 AM
> *To:* Myotoxin-II discussion <myotox at biophysics.uleth.ca>
> *Subject:* Re: [Myotox] myotoxin2-SDS titration
>
>
>
> Hi Everyone
>
> Looking at my notes from last time I prepared for the Shigemi
> I used 450 - 500ul to fill the tube, so I would say plan for 500ul.
>
> This means 10mg per nmr sample.
> --Mike
>
> *************************************************************************
> Michael Opyr
> Magnetic Resonance Facility Technician
> Faculty of Arts and Science
> The University of Lethbridge
> 4401 University Drive
> Lethbridge, Alberta, Canada T1K 3M4
> Phone: 1-403-332-4526
>
> -----Original Message-----
> From: Myotox <myotox-bounces at biophysics.uleth.ca> On Behalf Of Borries
> Demeler
> Sent: March 21, 2021 5:50 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.
>
>
> Thanks, Tony! We'll shoot for 20 mg/ml.
>
> Amy:
> please confirm if we have the required amounts available to do both the
> AUC and the NMR experiments. If not, please alert Bruno so he can send some
> more this week.
>
> Thanks!  -Borries
>
> On Sun, Mar 21, 2021 at 10:37:32PM +0000, Montina, Tony wrote:
> > 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:
> >
> > 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.
> >
> >
> > 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 <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
> >
> >
> >
> > _______________________________________________
> >
> > Myotox mailing list
> >
> > Myotox at biophysics.uleth.ca<mailto: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<mailto:bruno.lomonte at ucr.ac.cr>
> >
> > _______________________________________________
> > Myotox mailing list
> > Myotox at biophysics.uleth.ca
> > https://demeler7.uleth.ca/mailman/listinfo/myotox
> >
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> _______________________________________________
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>
> --
> Bruno Lomonte, Ph.D.
> Instituto Clodomiro Picado
> Universidad de Costa Rica
> San José, 11501
> COSTA RICA
> bruno.lomonte at ucr.ac.cr
> tel. +506  2511 7888
> cel. +506  8392 0012
>
>
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