[Myotox] SDS AUC update
Borries Demeler
demeler at gmail.com
Mon May 11 16:14:35 MDT 2020
This is great news! Thanks, Tony and Mike!
-Borries
On Mon, May 11, 2020 at 09:26:09PM +0000, Montina, Tony wrote:
> Hello everyone,
> Mike Opyr is preparing this sample on Thursday morning and we have the instrument booked until Monday morning.
> We hope to complete the 1D 1H, 2D COSY, 2D TOCSY, and possibly 2D selective HSQC over the weekend.
> The plan would then be to run the diffusion and relaxation measurements the following week.
> Stay tuned, I think we can have the NMR work done in the next couple of weeks.
> 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> On Behalf Of Hazendonk, Paul
> Sent: May 10, 2020 12:06 PM
> To: Myotoxin-II discussion <myotox at biophysics.uleth.ca>
> Subject: Re: [Myotox] SDS AUC update
>
> Depending on S/N and instrument availability this should be possible. The diffusion constant and relaxation rates should in principle be able to corroborate the monomer vs dimer conclusion.
> Sent from Outlook Mobile<https://aka.ms/blhgte>
>
> ________________________________
> From: Myotox <myotox-bounces at biophysics.uleth.ca<mailto:myotox-bounces at biophysics.uleth.ca>> on behalf of Borries Demeler <demeler at gmail.com<mailto:demeler at gmail.com>>
> Sent: Sunday, May 10, 2020 7:44:34 AM
> To: Myotoxin-II discussion <myotox at biophysics.uleth.ca<mailto:myotox at biophysics.uleth.ca>>
> Subject: Re: [Myotox] SDS AUC update
>
> 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. Please forward suspicious emails to phishing at uleth.ca<mailto:phishing at uleth.ca>.
>
> Hi Paul,
> this would be nice, any chance it can be done within the next couple of weeks?
> I am working on the SDS AUC results and looking through the comments.
> Will have another version ready for everyone to look at soon.
> Thanks, -Borries
>
>
>
> On Sat, May 09, 2020 at 08:58:57PM +0000, Hazendonk, Paul wrote:
> > We may be able to get away with a 1D and homonuclear 2Ds on 1H, a 1H DOSY and T1s and T2s of some strategically chosen methyl signals. Depending on SN this need not very long. I found some 1H assignments done in the mid nineties that should help.
> >
> > Sent from Outlook Mobile<https://aka.ms/blhgte>
> >
> > ________________________________
> > From: Myotox <myotox-bounces at biophysics.uleth.ca<mailto:myotox-bounces at biophysics.uleth.ca>> on behalf of Borries Demeler <demeler at gmail.com<mailto:demeler at gmail.com>>
> > Sent: Saturday, May 9, 2020 1:47:17 PM
> > To: Myotoxin-II discussion <myotox at biophysics.uleth.ca<mailto:myotox at biophysics.uleth.ca>>
> > Subject: Re: [Myotox] SDS AUC update
> >
> > 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. Please forward suspicious emails to phishing at uleth.ca<mailto:phishing at uleth.ca>.
> >
> > Thanks for the feedback, Tony, please keep us posted. I don't know how
> > long it takes, but if you can obtain useful data we'd rather include it.
> >
> > Thanks! -Borries
> >
> > On Sat, May 09, 2020 at 03:54:09PM +0000, Montina, Tony wrote:
> > > Hello all,
> > > Amy did provide Mike and I with the myotoxin a few weeks ago, but we have not had the chance to collect this data due to some instrument issues with the 700 MHz instrument and also the covid-19 situation. I completely understand if you must proceed without us for the manuscript.
> > > I will contact Michael Opyr and see if he can start data collection early next week.
> > > 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<mailto:myotox-bounces at biophysics.uleth.ca>> On Behalf Of Borries Demeler
> > > Sent: May 9, 2020 9:48 AM
> > > To: Myotoxin-II discussion <myotox at biophysics.uleth.ca<mailto:myotox at biophysics.uleth.ca>>
> > > Subject: Re: [Myotox] SDS AUC update
> > >
> > > 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. Please forward suspicious emails to phishing at uleth.ca<mailto:phishing at uleth.ca>.
> > >
> > > On Fri, May 08, 2020 at 06:34:49PM -0300, Ana Gisele da Costa Neves Ferreira wrote:
> > > > Dear Borries and colleagues,
> > > > I hope this email finds you and your families well, safe from COVID-19.
> > > > Currently, our lab team in Brazil is almost exclusively working from home.
> > > > Thinking about pending manuscripts, I was wondering whether you have
> > > > had the chance to generate new AUC/NMR data on myotoxin II before the pandemic.
> > >
> > > Hi Ana,
> > > Yes, our last AUC data was the run Amy did with myotoxin in the presence of SDS which conclusively showed that myotoxin dimerizes in the presence of SDS, and that this was apparently reversible/mass action dependent, if I recall correctly - need to review the data. Amy also provided myotoxin to Tony and Paul, but I am not sure what the status is on this, can you guys fill us in?
> > >
> > > > Anyway, I think we already have a very interesting set of results and
> > > > we should think about submitting a first manuscript. We also have nice
> > > > structural data on the complex made of myotoxin II and the inhibitor
> > > > DM64 that needs to be published ASAP (following the myotoxin paper).
> > > > Please let me know what you think.
> > >
> > > Bruno started the manuscript and we should all review where we are and get feedback from Paul to see if there is any NMR data that could be included, if not, I agree that it has been a long time in the making and we should proceed to publishing now. I think we have enough for a publication now, I would just like to know if it can be made strong by NMR data. So, Paul, can you please comment? Also, who has the latest version that we could all review?
> > >
> > > -Borries
> > >
> > >
> > >
> > > > Best regards,
> > > > AnaG
> > > >
> > > > Em sex., 13 de mar. de 2020 às 23:45, Hazendonk, Paul <
> > > > paul.hazendonk at uleth.ca<mailto:paul.hazendonk at uleth.ca>> escreveu:
> > > >
> > > > > I think relaxation dispersion experiments should be able to shed
> > > > > some light on this.
> > > > >
> > > > > Get Outlook for Android <https://aka.ms/ghei36>
> > > > >
> > > > > ------------------------------
> > > > > *From:* Myotox <myotox-bounces at biophysics.uleth.ca<mailto:myotox-bounces at biophysics.uleth.ca>> on behalf of
> > > > > Bruno Lomonte <bruno.lomonte at ucr.ac.cr<mailto:bruno.lomonte at ucr.ac.cr>>
> > > > > *Sent:* Friday, March 13, 2020 8:37:19 PM
> > > > > *To:* myotox at biophysics.uleth.ca<mailto:myotox at biophysics.uleth.ca> <myotox at biophysics.uleth.ca<mailto:myotox at biophysics.uleth.ca>>
> > > > > *Subject:* Re: [Myotox] SDS AUC update
> > > > >
> > > > >
> > > > > Fantastic that the paper helped Borries. And great that the results
> > > > > fitted well!
> > > > >
> > > > > As you know, I feel a bit overwhelmed by these biophysical
> > > > > calculations and techniques, because of my different background, so
> > > > > I have little to say. But at the same time I feel so impressed for
> > > > > all the information that these methods can provide! just great!
> > > > >
> > > > > Good luck in the next round of tests. If you succeed altogether to
> > > > > define the behaviour of this K49 protein regarding the monomer/dimer
> > > > > state 'dilemma' in solution, it will certainly be an important step
> > > > > forward and contribution to better understand its mechanism of action on cell membranes.
> > > > >
> > > > > Congratulations, and best regards to all
> > > > >
> > > > > Bruno
> > > > >
> > > > >
> > > > > +++
> > > > >
> > > > >
> > > > >
> > > > > On 3/13/2020 5:07 PM, Borries Demeler wrote:
> > > > >
> > > > > Bruno,
> > > > > this paper answered many of my questions, thank you very much for
> > > > > forwarding this. I would be curious if Paul's group could comment on
> > > > > what portions of these findings could be confirmed with NMR.
> > > > >
> > > > > Meanwhile, I have refined our AUC analysis of the SDS results further.
> > > > > Attached is a plot of a genetic algorithm/Monte Carlo analysis that
> > > > > shows the s-value distributions from the 4 experiments Amy ran. The
> > > > > high concentration protein + 1% SDS shows a peak that seems to
> > > > > suggest an oligomerization endpoint with s=3.1791e-13 s and D=8.0428e-07.
> > > > > Molar mass interpretations require knowledge of partial specific
> > > > > volume, which is discussed in the Uchiyama paper.
> > > > >
> > > > > Based on some of the estimates of vbar for SDS (0.87 ml/g) and the
> > > > > molar mass of an SDS monomer (~288 Da), it is now possible to
> > > > > calculate the ratio of SDS to myotoxin that results in a molar mass
> > > > > and vbar that is consistent with the hydrodynamic measurements. Only
> > > > > one ratio of protein and SDS will give a match. I wrote a small
> > > > > program to find that (using the vbar of SDS cited in the Uchiyama
> > > > > paper) and determined the protein MUST be dimeric and the number of
> > > > > SDS molecules must be 55 (tested between 0-70 bound SDS molecules).
> > > > > There is no other solution to this equation (see attached plots of monomer, dimer and trimer scenarios).
> > > > >
> > > > > The obtained difference in molar mass is 13 dalton at the match point.
> > > > > The attached images show the overlays of sum of molar masses, and
> > > > > the molar mass obtained from the Svedberg equation when we use the
> > > > > theoretical vbar from the combination of protein and SDS.
> > > > > Furthermore, the 55 molecules of SDS agrees very well with
> > > > > Uchiyama's paper, and the vbar at this point is 0.779 ml/g, very
> > > > > close to the measured value reported by Uchiyama. These numbers are
> > > > > dependent on the assumption that the vbar of SDS is 0.87 ml/g
> > > > >
> > > > > Therefore, I conclude that this larger species must be a dimer, and
> > > > > not a monomer or trimer.
> > > > >
> > > > > Going forward, Amy will measure the vbar using D2O density matching
> > > > > experiments on the dimer at a slightly higher protein concentration
> > > > > to push everything solidly to the dimer, and also measure at very
> > > > > low concentration so we can see pure monomer in SDS and measure the
> > > > > number of SDS molecules bound at that point. Based on these data we
> > > > > may even be able to measure Kds for this association in the presence of SDS.
> > > > >
> > > > > I like it when data make good sense!
> > > > >
> > > > > If it were worthwhile, we could also do the fluorescence anisotropy
> > > > > measurements in Missoula, where we have access to a very good FL
> > > > > spectroscopy lab. Let me know if this would be of interest.
> > > > > At this point I would be most interested in the information we could
> > > > > learn from NMR. Paul, Tony??
> > > > >
> > > > > -Borries
> > > > >
> > > > >
> > > > > On Thu, Mar 12, 2020 at 08:29:27PM -0600, Bruno Lomonte wrote:
> > > > >
> > > > > I see Borries... agree
> > > > >
> > > > > I found this 2007 paper that perhaps gives us important clues on
> > > > > this phenomenon of SDS-toxin interaction?
> > > > >
> > > > > the authors put forward the idea that SDS 'simulates' the effect of
> > > > > phospholipid interactions on the protein, and that this would have
> > > > > relevance to the membrane-damaging mechanism
> > > > >
> > > > > please see attached
> > > > >
> > > > > Bruno
> > > > >
> > > > >
> > > > > ++++
> > > > >
> > > > >
> > > > >
> > > > > On 3/12/2020 8:12 PM, Borries Demeler wrote:
> > > > >
> > > > > On Thu, Mar 12, 2020 at 07:26:21PM -0600, Bruno Lomonte wrote:
> > > > >
> > > > > Dear colleagues
> > > > >
> > > > >
> > > > > thanks for running these new experiments, and finding such
> > > > > interesting results!
> > > > >
> > > > >
> > > > > so this dimerization induced by SDS would explain the appearance of a smear
> > > > > around the dimeric value in such electrophoretic analyses? and this would
> > > > > support that under physiological conditions the toxin would be a
> > > > > monomer, the dimer being an artifact of crystallization perhaps? I
> > > > > am not sure if I am interpreting this correctly
> > > > >
> > > > > Dear Bruno,
> > > > > I don't think the interpretation is straightforward. SDS is having a
> > > > > strange effect on this protein, whose structural basis should be
> > > > > understood. Uchiyama's group found the same issue on their protein.
> > > > > Other than these two proteins I have never heard that SDS is causing
> > > > > proteins to dimerize. Furthermore, Myotoxin-II is highly soluble
> > > > > even at very high concentrations, it doesn't need SDS to be in
> > > > > solution. ALL proteins I know of *monomerize* when mixed with SDS,
> > > > > at least on SDS-PAGE gels. That's the exact opposite of what we are
> > > > > seeing here. SDS is a strong denaturant.
> > > > >
> > > > > I think the questions we need to answer is this:
> > > > > 1. why is the protein oligomerizing with an apparent end state
> > > > > (dimer or
> > > > > trimer?) when exposed to SDS? What is the structural basis for this?
> > > > >
> > > > > 2. is this behavior also observed when the protein is mixed with
> > > > > other lipids?
> > > > >
> > > > > 3. is this behavior somehow related to its toxic action, perhaps
> > > > > disrupting membranes?
> > > > >
> > > > > 4. What does the protein do when/if it binds to membranes?
> > > > >
> > > > > The Kd for oligomerization appears to be quite low. We will
> > > > > investigate this further by running in 1% (thanks, Amy, for the
> > > > > correction!) SDS again, but this time at lower concentration to see
> > > > > if it can push it to pure monomer in SDS. The process of
> > > > > oligomerization appears to be completely reversible and mass action driven.
> > > > >
> > > > >
> > > > > exciting news indeed
> > > > >
> > > > > Yes, fascinating. Susumu Uchiyama is a good friend of mine, I didn't
> > > > > even realize he was on the paper until today, he probably did the
> > > > > mass spec and AUC analysis, which, by the way, was well done. I will
> > > > > ask him if he has any further insights into the action of SDS.
> > > > >
> > > > > Regards, -Borries
> > > > > _______________________________________________
> > > > > Myotox mailing
> > > > > listMyotox at biophysics.uleth.cahttp://demeler7.uleth.ca/mailman/listi<mailto:listMyotox at biophysics.uleth.cahttp://demeler7.uleth.ca/mailman/listi>
> > > > > nfo/myotox
> > > > >
> > > > > --
> > > > > Bruno Lomonte, Ph.D.
> > > > > Instituto Clodomiro Picado
> > > > > Universidad de Costa Rica
> > > > > San José, 11501
> > > > > COSTA RICA
> > > > > bruno.lomonte at ucr.ac.cr<mailto:bruno.lomonte at ucr.ac.cr>
> > > > > tel. +506 2511 7888
> > > > > cel. +506 8392 0012
> > > > >
> > > > >
> > > > > _______________________________________________
> > > > > Myotox mailing
> > > > > listMyotox at biophysics.uleth.cahttp://demeler7.uleth.ca/mailman/listi<mailto:listMyotox at biophysics.uleth.cahttp://demeler7.uleth.ca/mailman/listi>
> > > > > nfo/myotox
> > > > >
> > > > >
> > > > > _______________________________________________
> > > > > Myotox mailing
> > > > > listMyotox at biophysics.uleth.cahttp://demeler7.uleth.ca/mailman/listi<mailto:listMyotox at biophysics.uleth.cahttp://demeler7.uleth.ca/mailman/listi>
> > > > > nfo/myotox
> > > > >
> > > > > --
> > > > > Bruno Lomonte, Ph.D.
> > > > > Instituto Clodomiro Picado
> > > > > Universidad de Costa Rica
> > > > > San José, 11501
> > > > > COSTA RICA
> > > > > bruno.lomonte at ucr.ac.cr<mailto:bruno.lomonte at ucr.ac.cr>
> > > > > tel. +506 2511 7888
> > > > > cel. +506 8392 0012
> > > > >
> > > > >
> > > > > _______________________________________________
> > > > > Myotox mailing list
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> > > > > http://demeler7.uleth.ca/mailman/listinfo/myotox
> > > > >
> > > >
> > > >
> > > > --
> > > > Ana Gisele da C. Neves Ferreira
> > > > Pesquisadora Titular em Saúde Pública
> > > > Chefe do Laboratorio de Toxinologia
> > > > Instituto Oswaldo Cruz - Fiocruz
> > > > Pavilhao Ozorio de Almeida, sala 05
> > > > Av. Brasil, 4365 - Manguinhos
> > > > Rio de Janeiro - RJ
> > > > 21040-900 Brasil
> > > > (+5521) 2562-1381 / 98801-5726
> > >
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> > >
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