[Myotox] myotoxin2-SDS titration

Tue Apr 20 15:42:30 MDT 2021

Wow, that's very close to what we estimated from AUC! Can you differentiate
between the uncomplexed myotoxin-II and that which is associated with SDS,
or is that the 9:1 ratio you are referring to? Either case, our AUC
analysis shows about 90% uncomplexed.
-Borries

On Tue, Apr 20, 2021 at 3:02 PM Hazendonk, Paul <paul.hazendonk at uleth.ca>
wrote:

> The line fitting puts the ration at about 9 SDS :1 MT11.
>
> -----Original Message-----
> From: Myotox <myotox-bounces at biophysics.uleth.ca> On Behalf Of Hazendonk,
> Paul
> Sent: April 13, 2021 5:50 PM
> To: Myotoxin-II discussion <myotox at biophysics.uleth.ca>
> Subject: Re: [Myotox] myotoxin2-SDS titration
>
> If that were the case I would expect to see sharp peaks corresponding to
> the beta, gamma and mainchain signals.  The DOSY and relaxation data will
> help sort it out.
>
> -----Original Message-----
> From: Myotox <myotox-bounces at biophysics.uleth.ca> On Behalf Of Borries
> Demeler
> Sent: April 13, 2021 5:47 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.
>
>
> It is good that we are seeing significant changes. Are the signals pretty
> distinct and not washed out from background SDS? Also, I would be curious
> if the SDS signals vary, indicating that there are multiple
> conformations/arrangements possible, or are they always looking similar?
> I would expect to see signals that are consistent with unbound myotoxin
> and also bound myotoxin.
> -Borries
>
> On Tue, Apr 13, 2021 at 10:27:17PM +0000, Paul Hazendonk wrote:
> > We should see those as well.  I need to do deconvolution analysis to
> get  more accurate picture.  What is weird is that the methyl signal has
> moved from 0.7 to 1.0 ppm and the alpha-CH2 moved from 3.88 to 3.55 ppm.
> In unbound SDS the methyl signal never goes above .87 ppm and the alpha-CH2
> has not been seen lower than 4.07 ppm.  I am not able to unambiguously
> identify the beta, gamma, and mainchain signals yet.  What’s more the
> alpha-CH2 signal has the splitting pattern of an AA’-BB’ suggesting it is
> locked into trans configuration at the SO3 end.
> >
> > From: Myotox <myotox-bounces at biophysics.uleth.ca> On Behalf Of Borries
> > Demeler
> > Sent: April 13, 2021 4:15 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>.
> >
> > 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
> <mailto: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<mailto:myotox-bounces at biophysics.u
> > leth.ca>> On Behalf Of Henrickson, Amy
> > Sent: April 13, 2021 2:30 PM
> > To: Myotoxin-II discussion
> > <myotox at biophysics.uleth.ca<mailto: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<mailto:myotox-bounces at biophysics.u
> > leth.ca>> on behalf of Borries Demeler
> > <demeler at gmail.com<mailto:demeler at gmail.com>>
> > Sent: Tuesday, April 13, 2021 2:14 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>.
> >
> >
> > 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<mailto: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<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>.
> > >
> > > 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<mailto:
> paul.hazendonk at uleth.ca%3cmailto: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<mailto:myotox-bounces at biophysics.uleth.ca%3cmailto: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<mailto
> > > :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>.
> > >
> > >
> > > 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 biophysi
> > > > cs.uleth.ca<mailto:myotox-bounces at biophysics.uleth.ca%3cmailto:myo
> > > > tox-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<mail
> > > > to: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<mailto:phishing at uleth.ca<mailto: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%3cmailto:paul.hazendonk at uleth.ca>><mailto:
> paul.hazendonk at uleth.ca<mailto:paul.hazendonk at uleth.ca<mailto:
> 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 biophysi
> > > > cs.uleth.ca<mailto:myotox-bounces at biophysics.uleth.ca%3cmailto:myo
> > > > tox-bounces at biophysics.uleth.ca>><mailto:myotox-bounces at biophysics
> > > > .u<mailto:myotox-bounces at biophysics.u<mailto:myotox-bounces at biophy
> > > > sics.u%3cmailto:myotox-bounces at biophysics.u>>
> > > > leth.ca<http://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<mail
> > > > to:myotox at biophysics.uleth.ca%3cmailto:myotox at biophysics.uleth.ca>
> > > > ><mailto:myotox at biophysics.uleth.ca<mailto:myotox at biophysics.uleth
> > > > .ca<mailto:myotox at biophysics.uleth.ca%3cmailto:myotox at biophysics.u
> > > > leth.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:
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> 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
> > > > _______________________________________________
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