[Myotox] high temperature NMR experiments

Mon Sep 14 17:04:00 MDT 2020

Dear colleagues,
The plans to finish the first paper seem just fine. I hope we can publish
this interesting story ASAP !
Best regards,
Ana

Em ter., 1 de set. de 2020 às 16:33, Montina, Tony <tony.montina at uleth.ca>
escreveu:

> Hello Borries,
> I think we can simply run a blank sample of the SDS in buffer to determine
> where the peaks fall in the spectrum.
> Mike and Amy, can the two of you please work together to get this blank
> sample prepared, as well as the sample with SDS included with myotoxin?
> Cheers
> 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> On Behalf Of Borries
> Demeler
> Sent: September 1, 2020 1:24 PM
> To: myotox at biophysics.uleth.ca
> Subject: Re: [Myotox] high temperature NMR experiments
>
> 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.
>
>
> Hi Tony,
> Thanks so much for the update! I think the plan is really solid and will
> provide additional information. The linewidth data gathered from the two
> temperature should tell us if there are differences between +/- SDS, which
> will be highly useful for the first paper, and tell us if SDS is either
> stabilizing, destabilizing, or has no effect. I would expect
> *some* change, since SDS clearly causes dimerization. We may not need
> residue assignments, just knowing the general trends could already be
> useful.
>
> I am copying the entire list to see if anyone else has any thoughts on the
> two-temperature experiments. The fluorescence data Amy collected suggested
> temperature effects starting around 40C, so staying below 40C is in my
> opinion a good idea, since it would be non-physiological anyway.
>
> Once we get the cryo-probe there will be plenty of additional structural
> information that we could access from 2D experiments, and that should shed
> further light on this interesting story, so for now the 1D experiments are
> fully sufficient.
>
> One point I am not clear on: When we add SDS to the buffer, how to you
> correct for the additional signal - is there some sort of baseline
> subtraction you can make? Of course we don't really need to characterize
> the SDS molecules, but we do care about the effect it has on the protein
> structure, and any details that we can obtain that would explain the
> dimerization effect.
>
> Amy, can does Tony have enough sample from the lyophilized stock that
> Bruno sent us?
>
> Thanks! -Borries
>
>
>
>
> On Tue, Sep 01, 2020 at 07:07:35PM +0000, Montina, Tony wrote:
> > Hello Borries,
> > I just met with Mike to go over the next steps of the myotoxin work.
> > We have tried 2D NMR work for a while now and we have come to the
> conclusion that most of the 2D work (if not all of it) will require a
> cryoprobe to complete.
> > So this is what we have determined Mike will do next:
> >
> > (1) Mike will take the normal myotoxin sample and run this sample at
> both 10 degrees and 37 degrees. At each of these temperatures he will
> obtain the 1H proton NMR and the T1 and T2 data for the proton NMR. This
> should allow Paul to determine if there are relaxation or linewidth affects
> over that temperature range. Mike will start these experiments as soon as
> possible (this week probably).
> >
> > (2) SDS experiments - we had decided to run the myotoxin in SDS and
> obtain the 1H NMR spectra and the T1 and T2 values in SDS. Do we need this
> for the first paper? If so, Amy, can you please help Mike with determining
> how to prepare the myotoxin sample in SDS with buffer.
> >
> > So Mike will start on step one above as soon as possible. Amy and
> Borries, please let us know about step 2 and Mike and Amy can make
> arrangements to figure out the sample preparation.
> >
> > Let me know if you have any questions or concerns.
> > 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: Borries Demeler <demeler at gmail.com>
> > Sent: August 27, 2020 12:38 PM
> > To: Montina, Tony <tony.montina at uleth.ca>
> > Cc: Henrickson, Amy <amy.henrickson at uleth.ca>; Hazendonk, Paul
> > <paul.hazendonk at uleth.ca>; Opyr, Michael <michael.opyr at uleth.ca>
> > Subject: Re: high temperature NMR experiments
> >
> > 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.
> >
> >
> > Thanks, guys! Also, Amy did collect the high temperature unfolding data
> based on intrinsic trp fluorescence change, and we did see changes in
> conformation starting at around 45C, is this enough for the high
> temperature experiments you had envisioned? If you need higher
> temperatures, since the stability is not sufficient at higher temperatures,
> I think we would have to skip the higher temperature experiments.
> >
> > Amy, can you please coordinate with Mike? I believe we have lyophilized
> sample so you can dissolve in whatever buffer (D2O, H2O, pH, etc.) suitable
> for your experiments, though I prefer to keep things in PO4 buffer when
> possible so we keep things comparable to the AUC experiments.
> >
> > Thanks, -Borries
> >
> >
> >
> > On Thu, Aug 27, 2020 at 06:28:25PM +0000, Montina, Tony wrote:
> > > Hello Borries and Amy,
> > > I was away for the second and third week of August for holidays and
> Mike was away this past week.
> > > Mike and I will meet on Monday or Tuesday next week, review our notes
> from the last meeting and discuss the next best steps.
> > > In the meantime, Amy, can you please make arrangements with Mike to
> drop off the sample to him?
> > > I believe he will be on campus tomorrow afternoon (Friday).
> > > Talk soon
> > > Tony
> > >
> > > Tony Montina
> > > Director, Science Operations
> > > Director, Magnetic Resonance Facility Instructor, Department of
> > > Chemistry and Biochemistry The University of Lethbridge,
> > > 4401 University Drive West
> > > Lethbridge, Alberta, Canada, T1K 3M4
> > > Office: SA6214 Science Commons 1-403-394-3927
> > > Lab: SA6216 Science Commons 1-403-329-2230
> > >
> > > -----Original Message-----
> > > From: Borries Demeler <demeler at gmail.com>
> > > Sent: August 27, 2020 9:28 AM
> > > To: Henrickson, Amy <amy.henrickson at uleth.ca>
> > > Cc: Hazendonk, Paul <paul.hazendonk at uleth.ca>; Montina, Tony
> > > <tony.montina at uleth.ca>; Opyr, Michael <michael.opyr at uleth.ca>
> > > Subject: Re: high temperature NMR experiments
> > >
> > > 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.
> > >
> > >
> > > Paul/Tony,
> > > do you have any updates for us?
> > >
> > > Thanks, -Borries
> > >
> > >
> > > On Tue, Aug 25, 2020 at 09:30:15PM +0000, Henrickson, Amy wrote:
> > > > Hello Everyone,
> > > >
> > > > Have you been able to book anytime to do the next experiments?
> > > >
> > > > Best,
> > > >
> > > > Amy Henrickson
> > > >
> > > > ________________________________
> > > > From: Borries Demeler <demeler at gmail.com>
> > > > Sent: Tuesday, August 4, 2020, 12:58 p.m.
> > > > To: Hazendonk, Paul; Montina, Tony; Henrickson, Amy
> > > > Subject: high temperature NMR experiments
> > > >
> > > > 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.
> > > >
> > > >
> > > > Hey guys,
> > > > In our last meeting Paul mentioned that it would be desirable to
> > > > measure myotoxin-II at higher temperature to get additional
> > > > information. Amy performed a melting curve using fluorescence
> > > > detection and determined a stability curve - Amy, can you please
> > > > indicate the onset of the transition temperature where the protein
> > > > changed fluorescence, and where the middle of the transition is
> > > > for myotoxin in the presence and absence of SDS?
> > > >
> > > > I would like to know what experiments you would like to do next
> > > > and what information we could add to the paper from them. As we
> > > > discussed, this could turn into a rabbit hole of further
> > > > experiments, and some of them may be best postponed until we get a
> > > > cryo probe. For now, we just want to pick the low-hanging fruit,
> > > > and Amy obtained more sample at high purity from Costa Rica we can
> > > > now give to you. Please contact Amy to arrange for her to bring
> > > > them to you. The samples we have are lyophilized and can be
> suspended by you in any buffer needed.
> > > >
> > > > Please let us know how you want to proceed.
> > > >
> > > > Thanks! -Borries
> > > >
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>

-- 
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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