[Myotox] SDS AUC update

Mon May 11 16:20:14 MDT 2020

Ideed !!

Em seg., 11 de mai. de 2020 às 19:14, Borries Demeler <demeler at gmail.com>
escreveu:

> 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
> @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
> @biophysics.uleth.cahttp://demeler7.uleth.ca/mailman/listi>
> > > > > > nfo/myotox
> > > > > >
> > > > > >
> > > > > > _______________________________________________
> > > > > > Myotox mailing
> > > > > > listMyotox at biophysics.uleth.cahttp://
> demeler7.uleth.ca/mailman/listi<mailto:listMyotox
> @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
> > > > > > Myotox at biophysics.uleth.ca<mailto:Myotox at biophysics.uleth.ca>
> > > > > > 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
> > > >
> > > > > _______________________________________________
> > > > > Myotox mailing list
> > > > > Myotox at biophysics.uleth.ca<mailto:Myotox at biophysics.uleth.ca>
> > > > > http://demeler7.uleth.ca/mailman/listinfo/myotox
> > > >
> > > > _______________________________________________
> > > > Myotox mailing list
> > > > Myotox at biophysics.uleth.ca<mailto:Myotox at biophysics.uleth.ca>
> > > > http://demeler7.uleth.ca/mailman/listinfo/myotox
> > > > _______________________________________________
> > > > Myotox mailing list
> > > > Myotox at biophysics.uleth.ca<mailto:Myotox at biophysics.uleth.ca>
> > > > http://demeler7.uleth.ca/mailman/listinfo/myotox
> > > _______________________________________________
> > > Myotox mailing list
> > > Myotox at biophysics.uleth.ca<mailto:Myotox at biophysics.uleth.ca>
> > > http://demeler7.uleth.ca/mailman/listinfo/myotox
> >
> > > _______________________________________________
> > > Myotox mailing list
> > > Myotox at biophysics.uleth.ca<mailto:Myotox at biophysics.uleth.ca>
> > > http://demeler7.uleth.ca/mailman/listinfo/myotox
> >
> > _______________________________________________
> > Myotox mailing list
> > Myotox at biophysics.uleth.ca<mailto:Myotox at biophysics.uleth.ca>
> > http://demeler7.uleth.ca/mailman/listinfo/myotox
>
> > _______________________________________________
> > Myotox mailing list
> > Myotox at biophysics.uleth.ca
> > http://demeler7.uleth.ca/mailman/listinfo/myotox
>
> _______________________________________________
> Myotox mailing list
> Myotox at biophysics.uleth.ca
> 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
-------------- next part --------------
An HTML attachment was scrubbed...
URL: <http://demeler7.uleth.ca/pipermail/myotox/attachments/20200511/14d9ca16/attachment-0001.html>