[Myotox] myotoxin2-SDS titration

Sat Apr 24 16:39:00 MDT 2021

I just finished the line fitting.  I think I found all the SDS signals.  The CH2 next to the SO3- shows severe strain forcing it to be planar.  This can be seen as the triplet turns into what looks like a quartet, however that is deceiving as it is what it referred to as an AB quartet.  The couplings and the inequivalence between the protons prove the distorted nature of this carbon.  This means that there is a very strong binding interaction on the SO3- side, likely with the guanidinium groups of the arginine residues.   This jives with the stoichiometry I determined to be 5.5 SDS: 1 T2II.   I am waiting for the analysis of the relaxation and DOSY data, which will help me confirm the assignment of the SDS signals, and that the binding takes place with the arginines.

From: Myotox <myotox-bounces at biophysics.uleth.ca> On Behalf Of Hazendonk, Paul
Sent: April 20, 2021 5:29 PM
To: Myotoxin-II discussion <myotox at biophysics.uleth.ca>
Subject: Re: [Myotox] myotoxin2-SDS titration

I see no free SDS.  It should show a triplet at 0.7-.9 ppm at 10% the intensity which we should be able to see. EXSY will be able to confirm that once we run the 2D’s.

From: Myotox <myotox-bounces at biophysics.uleth.ca<mailto:myotox-bounces at biophysics.uleth.ca>> On Behalf Of Borries Demeler
Sent: April 20, 2021 3:43 PM
To: Myotoxin-II discussion <myotox at biophysics.uleth.ca<mailto:myotox at biophysics.uleth.ca>>
Subject: Re: [Myotox] myotoxin2-SDS titration

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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<mailto: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<mailto: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<mailto: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<mailto: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<mailto:myotox at biophysics.uleth.ca>>
Subject: Re: [Myotox] myotoxin2-SDS titration

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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<mailto: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<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>>.
>
> 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><mailto: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.uleth.ca><mailto:myotox-bounces at biophysics.u<mailto:myotox-bounces at biophysics.u>
> leth.ca<http://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><mailto: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.uleth.ca><mailto:myotox-bounces at biophysics.u<mailto:myotox-bounces at biophysics.u>
> leth.ca<http://leth.ca>>> on behalf of Borries Demeler
> <demeler at gmail.com<mailto:demeler at gmail.com><mailto: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><mailto: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>>.
>
>
> 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><mailto:myotox-bounces at biophysics<mailto:myotox-bounces at biophysics>
> > .uleth.ca<http://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><mailto: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>><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<mailto:paul.hazendonk at uleth.ca><mailto:paul.hazendonk at uleth.ca<mailto:paul.hazendonk at uleth.ca>%3cmailto:paul.hazendonk at uleth.ca<mailto:3cmailto%3Apaul.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<mailto:myotox-bounces at biophysics>
> > .uleth.ca<http://uleth.ca><mailto:myotox-bounces at biophysics.uleth.ca<mailto:myotox-bounces at biophysics.uleth.ca>%3cmailto:myotox-
> > bounces at biophysics.uleth.ca<mailto: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<mailto:myotox at biophysics.uleth.ca><mailto
> > :myotox at biophysics.uleth.ca<mailto:myotox at biophysics.uleth.ca>%3cmailto:myotox at biophysics.uleth.ca<mailto:3cmailto%3Amyotox 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>>.
> >
> >
> > 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><mailto:myotox-bounces at biophysi<mailto:myotox-bounces at biophysi>
> > > cs.uleth.ca<http://cs.uleth.ca><mailto:myotox-bounces at biophysics.uleth.ca<mailto:myotox-bounces at biophysics.uleth.ca>%3cmailto:myo
> > > tox-bounces at biophysics.uleth.ca<mailto: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><mailto:myotox at biophysics.uleth.ca<mailto:myotox at biophysics.uleth.ca><mail
> > > to:myotox at biophysics.uleth.ca<mailto:to%3Amyotox at biophysics.uleth.ca>%3cmailto:myotox at biophysics.uleth.ca<mailto:3cmailto%3Amyotox 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>><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<mailto:phishing at uleth.ca>%3e%3cmailto:phishing at uleth.ca<mailto:3e%253cmailto%3Aphishing at uleth.ca>%3cmailto:phishing at uleth.ca<mailto:3cmailto%3Aphishing 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><mailto:paul.hazendonk at uleth.ca<mailto:paul.hazendonk at uleth.ca>%3cmailto:paul.hazendonk at uleth.ca<mailto:3cmailto%3Apaul.hazendonk at uleth.ca>>><mailto:paul.hazendonk at uleth.ca<mailto:paul.hazendonk at uleth.ca><mailto: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<mailto:3cmailto%3Apaul.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 biophysi<mailto:myotox-bounces at biophysi>
> > > cs.uleth.ca<http://cs.uleth.ca><mailto:myotox-bounces at biophysics.uleth.ca<mailto:myotox-bounces at biophysics.uleth.ca>%3cmailto:myo
> > > tox-bounces at biophysics.uleth.ca<mailto:tox-bounces at biophysics.uleth.ca>>><mailto:myotox-bounces at biophysics<mailto:myotox-bounces at biophysics>
> > > .u<mailto:myotox-bounces at biophysics.u<mailto:myotox-bounces at biophysics.u><mailto:myotox-bounces at biophy<mailto:myotox-bounces at biophy>
> > > sics.u%3cmailto:myotox-bounces at biophysics.u>>
> > > leth.ca<http://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><mailto:myotox at biophysics.uleth.ca<mailto:myotox at biophysics.uleth.ca><mail
> > > to:myotox at biophysics.uleth.ca<mailto:to%3Amyotox at biophysics.uleth.ca>%3cmailto:myotox at biophysics.uleth.ca<mailto:3cmailto%3Amyotox at biophysics.uleth.ca>>
> > > ><mailto:myotox at biophysics.uleth.ca<mailto:myotox at biophysics.uleth.ca><mailto:myotox at biophysics.uleth<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<http://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: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<mailto:phishing at uleth.ca><mailto:phishing at uleth.ca<mailto:phishing at uleth.ca>%3e%3cmailto:phishing at uleth.ca<mailto:3e%253cmailto%3Aphishing at uleth.ca>%3cmailto:phishing at uleth.ca<mailto:3cmailto%3Aphishing 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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