[Myotox] high temperature NMR experiments
Bruno Lomonte
bruno.lomonte at ucr.ac.cr
Fri Dec 4 16:06:30 MST 2020
Dear all,
Ana and Borries just read my mind... I was just going to ask the same
today! :)
Of course it would be totally understandable if it has been difficult to
run the assays, since obviously we are all facing the health risks of
this pandemy
Sending you all my best and hoping that everyone is doing well, science
aside
Bruno
+++++
On 12/4/2020 2:15 PM, Ana Gisele da Costa Neves Ferreira wrote:
> Dear colleagues,
> I hope you and your families are in good health. We are already at the
> end of the year and I think it would be great if we could finish the
> manuscript on myotoxin II and submit it for publication as soon as
> possible. Could you give us an update on the latest NMR data progress ?
> I wish you a joyful and peaceful holiday season, despite these
> difficult times.
> Warm regards,
> Ana Gisele
>
> Em ter., 1 de set. de 2020 às 16:33, Montina, Tony
> <tony.montina at uleth.ca <mailto: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
> <mailto:myotox-bounces at biophysics.uleth.ca>> On Behalf Of Borries
> Demeler
> Sent: September 1, 2020 1:24 PM
> To: myotox at biophysics.uleth.ca <mailto: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 <mailto: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 <mailto:demeler at gmail.com>>
> > Sent: August 27, 2020 12:38 PM
> > To: Montina, Tony <tony.montina at uleth.ca
> <mailto:tony.montina at uleth.ca>>
> > Cc: Henrickson, Amy <amy.henrickson at uleth.ca
> <mailto:amy.henrickson at uleth.ca>>; Hazendonk, Paul
> > <paul.hazendonk at uleth.ca <mailto:paul.hazendonk at uleth.ca>>;
> Opyr, Michael <michael.opyr at uleth.ca <mailto: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 <mailto: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
> <mailto:demeler at gmail.com>>
> > > Sent: August 27, 2020 9:28 AM
> > > To: Henrickson, Amy <amy.henrickson at uleth.ca
> <mailto:amy.henrickson at uleth.ca>>
> > > Cc: Hazendonk, Paul <paul.hazendonk at uleth.ca
> <mailto:paul.hazendonk at uleth.ca>>; Montina, Tony
> > > <tony.montina at uleth.ca <mailto:tony.montina at uleth.ca>>; Opyr,
> Michael <michael.opyr at uleth.ca <mailto: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 <mailto: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
> <mailto: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 <mailto: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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> Myotox at biophysics.uleth.ca
> https://demeler7.uleth.ca/mailman/listinfo/myotox
--
Bruno Lomonte, Ph.D.
Instituto Clodomiro Picado
Facultad de Microbiología
Universidad de Costa Rica
San José, COSTA RICA
tel.of. (+506) 2511 7888
bruno.lomonte at ucr.ac.cr
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