[Myotox] myotoxin2-SDS titration

Tue Apr 13 16:14:50 MDT 2021

That is very reasonable considering some of the myotoxin molecules are
likely not bound to SDS, and is definitely in the same ballpark as what we
were measuring.
-Borries

On Tue, Apr 13, 2021 at 4:12 PM Hazendonk, Paul <paul.hazendonk at uleth.ca>
wrote:

> The integration data suggest somewhere between 2-4:1 ration SDS:Toxin.
>
>
>
> *From:* Myotox <myotox-bounces at biophysics.uleth.ca> *On Behalf Of *Henrickson,
> Amy
> *Sent:* April 13, 2021 2:30 PM
> *To:* Myotoxin-II discussion <myotox at biophysics.uleth.ca>
> *Subject:* Re: [Myotox] myotoxin2-SDS titration
>
>
>
> Hello,
>
>
>
> It should also be mentioned that the AUC sample was 20 fold diluted from
> the sample that was sent for NMR. Therefore if there is a mass action
> effect, the sample measured in the NMR maybe different from the results
> seen in Borries's attached image.
>
>
>
> Cheers,
>
> Amy H
> ------------------------------
>
> *From:* Myotox <myotox-bounces at biophysics.uleth.ca> on behalf of Borries
> Demeler <demeler at gmail.com>
> *Sent:* Tuesday, April 13, 2021 2:14 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.
>
>
> Hi Paul,
>
> Amy checked the sample we sent for NMR analysis. It wasn't quite as high
> in concentration since we were trying to titrate the SDS and check our
> results, which required further sample, so we ran somewhat low.
>
> Anyway, what you have has a limited amount of SDS in it which avoids
> aggregation and makes about 10% of complex (6 or 7-mer, as best as I can
> tell). Everything is in D2O.
>
> I would expect that you see a mixture of signals. The mixture will be
> from uncomplexed myotoxin giving you pure signals from myotoxin, and
> then from myotoxin molecules that are complexed with SDS. THere should
> not be much, if any free SDS present. So the background from free SDS
> should be virtually eliminated.
>
> What I don't know is if the amount of signal you get from the
> SDS/Myotoxin interaction is strong enough to get good information. If
> not, we may have to repeat it with more SDS present. It is just very
> difficult to estimate the amount of SDS needed when you change the
> protein concentration since you are guessing essentially on both.
> It *looks* like 7:1 SDS:Myotoxin, but I am not sure. Perhaps the NMR can
> provide further clarity, though there will be a mixture of free and
> complexed Myotoxin present. I felt it may be best to limit the amount of
> complex and err on the side of too little SDS rather than the opposite,
> which gives non-specific aggregation and probably overpowering signal
> from free SDS.
>
> If we have to do this again to get a clearer picture, we will need quite
> a bit more sample (sorry, Bruno), but maybe its not necessary and Paul
> can figure it out from here.
>
> Let me know if you have any thoughts or comments. The picture of the NMR
> sample's AUC result is attached.
>
> Regards, -Borries
>
>
>
>
> On Sun, Apr 11, 2021 at 09:48:41PM +0000, Paul Hazendonk wrote:
> > The SDS chemical shifts have moved all over the place.  It will take mw
> some time to confirm the assignments of all the SDS signals, however it
> definitely looks like there is an interesting story to tell.
> >
> > From: Myotox <myotox-bounces at biophysics.uleth.ca> On Behalf Of Borries
> Demeler
> > Sent: April 1, 2021 10:01 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<
> mailto:phishing at uleth.ca <phishing at uleth.ca>>.
> >
> > AUC is not a choice method for studying kinetics since the time scale of
> sedimentation is too slow, except for very large molecules and high rotor
> speeds. Myotoxin is only 13.5 kDa or so, therefore there isn't any kinetics
> you could pick up. But you can measure Kds very well, which is what we are
> doing now. I'll explain more on Tuesday.
> > -b.
> >
> > On Thu, Apr 1, 2021 at 6:16 PM Hazendonk, Paul <
> paul.hazendonk at uleth.ca<mailto:paul.hazendonk at uleth.ca>> wrote:
> > So if you have association-dissociation taking place, the slope at the
> inflection point does not change with speed.  I guess the exchange rates
> are too high.  How about for very slow processes?  I guess at that point
> you would see separate inflections.
> >
> > -----Original Message-----
> > From: Myotox <
> myotox-bounces at biophysics.uleth.ca<mailto:myotox-bounces at biophysics.uleth.ca>>
> On Behalf Of Borries Demeler
> > Sent: April 1, 2021 5:42 PM
> > To: Myotoxin-II discussion <
> myotox at biophysics.uleth.ca<mailto: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>>.
> >
> >
> > Varying the speed only affects the resolution:
> >
> > high speed: good resolution on s, poor resolution on D low speed: good
> resolution on D, poor resolution on s.
> >
> > -Borries
> >
> > On Thu, Apr 01, 2021 at 11:16:34PM +0000, Paul Hazendonk wrote:
> > > Can you do dynamic studies by varying the speed of centrifugation?
> I.e. the slope as a function of speed?
> > >
> > > From: Myotox <
> myotox-bounces at biophysics.uleth.ca<mailto:myotox-bounces at biophysics.uleth.ca>>
> On Behalf Of Borries
> > > Demeler
> > > Sent: April 1, 2021 4:37 PM
> > > To: Myotoxin-II discussion <
> myotox at biophysics.uleth.ca<mailto: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><mailto:phishing at uleth.ca<mailto:phishing at uleth.ca
> <phishing at uleth.ca%3e%3cmailto:phishing at uleth.ca%3cmailto:phishing at uleth.ca>
> >>.
> > >
> > > The s-value is reflective of the size of the molecule. A 5.5 s species
> is much bigger than a 2 s species. The y axis tells you the relative amount
> of species with a given s-value. It is a standard integral sedimentation
> distribution. I am happy to have a zoom meeting to discuss further these
> interesting trends.
> > > -Borries
> > >
> > > On Thu, Apr 1, 2021 at 4:20 PM Hazendonk, Paul <
> paul.hazendonk at uleth.ca<mailto:paul.hazendonk at uleth.ca><
> mailto:paul.hazendonk at uleth.ca<mailto:paul.hazendonk at uleth.ca
> <paul.hazendonk at uleth.ca%3cmailto:paul.hazendonk at uleth.ca>>>> wrote:
> > > What is the best way to interpret these curves.  I have not seen
> ultracentrifugation data since 1991.  Should we be looking at the position
> of the inflection point?
> > >
> > >
> > > -----Original Message-----
> > > From: Myotox
> > > <
> myotox-bounces at biophysics.uleth.ca<mailto:myotox-bounces at biophysics.uleth.ca
> ><mailto:myotox-bounces at biophysics.u<mailto:myotox-bounces at biophysics.u
> <myotox-bounces at biophysics.u%3cmailto:myotox-bounces at biophysics.u>>
> > > leth.ca<http://leth.ca>>> On Behalf Of Borries Demeler
> > > Sent: April 1, 2021 11:57 AM
> > > To: Myotoxin-II discussion
> > > <myotox at biophysics.uleth.ca<mailto:myotox at biophysics.uleth.ca><
> mailto:myotox at biophysics.uleth.ca<mailto:myotox at biophysics.uleth.ca
> <myotox at biophysics.uleth.ca%3cmailto: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><mailto:phishing at uleth.ca<mailto:phishing at uleth.ca
> <phishing at uleth.ca%3e%3cmailto:phishing at uleth.ca%3cmailto:phishing at uleth.ca>
> >>.
> > >
> > >
> > > Dear all,
> > > we have results back from the myotoxin-2 SDS titration experiments in
> AUC. I feel these experiments provided some very interesting results and
> are probably the most informative ones we have done so far. I also think
> that they will be extremely helpful in designing the *correct* NMR
> experiment. I went through the effort to write this up with figures, please
> review carefully the attached document and read the explanations I believe
> explain these results (which were quite unintuitive at first, but now make
> sense to me). Then let's schedule another meeting and discussion of next
> steps.
> > >
> > > -Borries
> > > _______________________________________________
> > > Myotox mailing list
> > >
> Myotox at biophysics.uleth.ca<mailto:Myotox at biophysics.uleth.ca><mailto:Myotox at biophysics.uleth.ca<mailto:Myotox at biophysics.uleth.ca
> >>
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> >
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