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Hello, </div>
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<br>
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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. </div>
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<br>
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Cheers, </div>
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Amy H</div>
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<div id="divRplyFwdMsg" dir="ltr"><font face="Calibri, sans-serif" style="font-size:11pt" color="#000000"><b>From:</b> Myotox <myotox-bounces@biophysics.uleth.ca> on behalf of Borries Demeler <demeler@gmail.com><br>
<b>Sent:</b> Tuesday, April 13, 2021 2:14 PM<br>
<b>To:</b> Myotoxin-II discussion <myotox@biophysics.uleth.ca><br>
<b>Subject:</b> Re: [Myotox] myotoxin2-SDS titration</font>
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<div class="PlainText">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@uleth.ca.<br>
<br>
<br>
Hi Paul,<br>
<br>
Amy checked the sample we sent for NMR analysis. It wasn't quite as high<br>
in concentration since we were trying to titrate the SDS and check our<br>
results, which required further sample, so we ran somewhat low.<br>
<br>
Anyway, what you have has a limited amount of SDS in it which avoids<br>
aggregation and makes about 10% of complex (6 or 7-mer, as best as I can<br>
tell). Everything is in D2O.<br>
<br>
I would expect that you see a mixture of signals. The mixture will be<br>
from uncomplexed myotoxin giving you pure signals from myotoxin, and<br>
then from myotoxin molecules that are complexed with SDS. THere should<br>
not be much, if any free SDS present. So the background from free SDS<br>
should be virtually eliminated.<br>
<br>
What I don't know is if the amount of signal you get from the<br>
SDS/Myotoxin interaction is strong enough to get good information. If<br>
not, we may have to repeat it with more SDS present. It is just very<br>
difficult to estimate the amount of SDS needed when you change the<br>
protein concentration since you are guessing essentially on both.<br>
It *looks* like 7:1 SDS:Myotoxin, but I am not sure. Perhaps the NMR can<br>
provide further clarity, though there will be a mixture of free and<br>
complexed Myotoxin present. I felt it may be best to limit the amount of<br>
complex and err on the side of too little SDS rather than the opposite,<br>
which gives non-specific aggregation and probably overpowering signal<br>
from free SDS.<br>
<br>
If we have to do this again to get a clearer picture, we will need quite<br>
a bit more sample (sorry, Bruno), but maybe its not necessary and Paul<br>
can figure it out from here.<br>
<br>
Let me know if you have any thoughts or comments. The picture of the NMR<br>
sample's AUC result is attached.<br>
<br>
Regards, -Borries<br>
<br>
<br>
<br>
<br>
On Sun, Apr 11, 2021 at 09:48:41PM +0000, Paul Hazendonk wrote:<br>
> 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.<br>
><br>
> From: Myotox <myotox-bounces@biophysics.uleth.ca> On Behalf Of Borries Demeler<br>
> Sent: April 1, 2021 10:01 PM<br>
> To: Myotoxin-II discussion <myotox@biophysics.uleth.ca><br>
> Subject: Re: [Myotox] myotoxin2-SDS titration<br>
><br>
> 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@uleth.ca<<a href="mailto:phishing@uleth.ca">mailto:phishing@uleth.ca</a>>.<br>
><br>
> 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.<br>
> -b.<br>
><br>
> On Thu, Apr 1, 2021 at 6:16 PM Hazendonk, Paul <paul.hazendonk@uleth.ca<mailto:paul.hazendonk@uleth.ca>> wrote:<br>
> 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.<br>
><br>
> -----Original Message-----<br>
> From: Myotox <myotox-bounces@biophysics.uleth.ca<mailto:myotox-bounces@biophysics.uleth.ca>> On Behalf Of Borries Demeler<br>
> Sent: April 1, 2021 5:42 PM<br>
> To: Myotoxin-II discussion <myotox@biophysics.uleth.ca<mailto:myotox@biophysics.uleth.ca>><br>
> Subject: Re: [Myotox] myotoxin2-SDS titration<br>
><br>
> 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@uleth.ca<<a href="mailto:phishing@uleth.ca">mailto:phishing@uleth.ca</a>>.<br>
><br>
><br>
> Varying the speed only affects the resolution:<br>
><br>
> high speed: good resolution on s, poor resolution on D low speed: good resolution on D, poor resolution on s.<br>
><br>
> -Borries<br>
><br>
> On Thu, Apr 01, 2021 at 11:16:34PM +0000, Paul Hazendonk wrote:<br>
> > Can you do dynamic studies by varying the speed of centrifugation? I.e. the slope as a function of speed?<br>
> ><br>
> > From: Myotox <myotox-bounces@biophysics.uleth.ca<mailto:myotox-bounces@biophysics.uleth.ca>> On Behalf Of Borries<br>
> > Demeler<br>
> > Sent: April 1, 2021 4:37 PM<br>
> > To: Myotoxin-II discussion <myotox@biophysics.uleth.ca<mailto:myotox@biophysics.uleth.ca>><br>
> > Subject: Re: [Myotox] myotoxin2-SDS titration<br>
> ><br>
> > 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@uleth.ca<<a href="mailto:phishing@uleth.ca><mailto:phishing@uleth.ca<mailto:phishing@uleth.ca">mailto:phishing@uleth.ca><mailto:phishing@uleth.ca<mailto:phishing@uleth.ca</a>>>.<br>
> ><br>
> > 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.<br>
> > -Borries<br>
> ><br>
> > On Thu, Apr 1, 2021 at 4:20 PM Hazendonk, Paul <paul.hazendonk@uleth.ca<mailto:paul.hazendonk@uleth.ca><mailto:paul.hazendonk@uleth.ca<mailto:paul.hazendonk@uleth.ca>>> wrote:<br>
> > 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?<br>
> ><br>
> ><br>
> > -----Original Message-----<br>
> > From: Myotox<br>
> > <myotox-bounces@biophysics.uleth.ca<mailto:myotox-bounces@biophysics.uleth.ca><mailto:myotox-bounces@biophysics.u<mailto:myotox-bounces@biophysics.u><br>
> > leth.ca<<a href="http://leth.ca">http://leth.ca</a>>>> On Behalf Of Borries Demeler<br>
> > Sent: April 1, 2021 11:57 AM<br>
> > To: Myotoxin-II discussion<br>
> > <myotox@biophysics.uleth.ca<mailto:myotox@biophysics.uleth.ca><mailto:myotox@biophysics.uleth.ca<mailto:myotox@biophysics.uleth.ca>>><br>
> > Subject: Re: [Myotox] myotoxin2-SDS titration<br>
> ><br>
> > 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@uleth.ca<<a href="mailto:phishing@uleth.ca><mailto:phishing@uleth.ca<mailto:phishing@uleth.ca">mailto:phishing@uleth.ca><mailto:phishing@uleth.ca<mailto:phishing@uleth.ca</a>>>.<br>
> ><br>
> ><br>
> > Dear all,<br>
> > 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.<br>
> ><br>
> > -Borries<br>
> > _______________________________________________<br>
> > Myotox mailing list<br>
> > Myotox@biophysics.uleth.ca<mailto:Myotox@biophysics.uleth.ca><mailto:Myotox@biophysics.uleth.ca<mailto:Myotox@biophysics.uleth.ca>><br>
> > <a href="https://demeler7.uleth.ca/mailman/listinfo/myotox">https://demeler7.uleth.ca/mailman/listinfo/myotox</a><br>
><br>
> > _______________________________________________<br>
> > Myotox mailing list<br>
> > Myotox@biophysics.uleth.ca<mailto:Myotox@biophysics.uleth.ca><br>
> > <a href="https://demeler7.uleth.ca/mailman/listinfo/myotox">https://demeler7.uleth.ca/mailman/listinfo/myotox</a><br>
><br>
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