[Myotox] Samples for NMR
Borries Demeler
demeler at gmail.com
Fri Feb 21 15:08:18 MST 2020
Here is the mytoxin-II sequence:
0001 slfelgkmil qetgknpaks ygaygcncgv lgrgkpkdat drccyvhkcc ykkltgcnpk
0061 kdrysyswkd ktivcgenns clkelcecdk avaiclrenl ntynkkyryy lkplckkada
0121 c
-b.
On Fri, Feb 21, 2020 at 2:43 PM Hazendonk, Paul <paul.hazendonk at uleth.ca>
wrote:
> Borries could you send me the sequence.
>
> Get Outlook for Android <https://aka.ms/ghei36>
>
> ------------------------------
> *From:* Myotox <myotox-bounces at biophysics.uleth.ca> on behalf of Steele,
> Harmen <harmen.steele at umconnect.umt.edu>
> *Sent:* Friday, February 21, 2020 8:33:38 AM
> *To:* Myotoxin-II discussion <myotox at biophysics.uleth.ca>
> *Subject:* Re: [Myotox] Samples for NMR
>
> We can purchase a plethora of lipids from Avanti. This would allow up to
> mix together a combination that would make since as a model system. I think
> that we want to mimic the lipid composition in muscle? A quick Avanti
> search didn't return "pre-mixed" answer. Give me a little bit of time to
> Google and formulate a lipid mix that could be a good mimic.
>
>
> Best regards,
>
> Harmen Steele, Ph.D.
> Postdoctoral Researcher
> harmen.steele at umontana.edu
>
> +406 529 9669 - mobile
> Skype Me: harmensteele <callto://harmensteele/>
> Amateur Radio Call Sign: K9HBS
> I am a UM Ally <http://www.umt.edu/umallies/>
>
> This email was sent using 95% recycled binary bits and 99.9% recycled
> electrons.
>
>
> On 2/21/20, 8:12 AM, "Myotox on behalf of Montina, Tony" <
> myotox-bounces at biophysics.uleth.ca on behalf of tony.montina at uleth.ca>
> wrote:
>
> Hello all,
> I agree with Borries that all model systems should be run with AUC
> first. I also agree that Harmen makes some very good points.
> Reading that the CL lipid is only found in the mitochondrial matrix
> and the testes makes me think we should probably not use this as our model
> lipid system.
> As Borries asked, does anyone have any suggestions on how we could go
> about preparing or purchasing the muscle lipid mixture?
> Mike, Paul and I will continue with the myotoxin NMR work while we
> sort out this membrane decision.
> Cheers
> Tony
>
> Tony Montina
> Interim Director, Science Commons Academic 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: Myotox <myotox-bounces at biophysics.uleth.ca> On Behalf Of
> Borries Demeler
> Sent: February 21, 2020 8:01 AM
> To: myotox at biophysics.uleth.ca
> Subject: [Myotox] Samples for NMR
>
>
> I think we should have further discussions regarding the next steps
> regarding lipid systems to be studied by NMR. Harmen brings up some good
> points:
>
> 1. Nanodiscs or liposomes? There are pros and cons for each.
> 2. what type of lipid is most representative?
>
> I know Bruno mentioned that the type of lipid system doesn't seem to
> be so important, however, we may want to make some educated decisions on
> this.
> Myotoxin-II seems to be specific for muscle tissue, so perhaps we
> should try to purify some muscle membrane?
>
> I am not sure how much expertise we would have around here to help
> with this, but are there some commercially available lipids that could
> mimick muscle membrane? Suggestions?
>
> Finally, I suggest that once we settle on question 1 we should run all
> of these experiments with AUC controls to see what we may expect in an NMR
> experiment.
>
> Regards, -Borries
>
> ----- Forwarded message from "Steele, Harmen" <
> harmen.steele at umconnect.umt.edu> -----
>
> Date: Fri, 21 Feb 2020 14:41:06 +0000
> From: "Steele, Harmen" <harmen.steele at umconnect.umt.edu>
> Subject: Re: Samples for NMR
>
> The samples that I sent for NMR in August were 10 uM DMPC nanodisc in
> 50 mM deuterated Potassium Phosphate buffer at pH 7.0. There was
> additional buffer to dilute. Those samples should be OK with the exception
> of any deuteration that might have occurred.
>
> While I agree that ND are a good model system. I’m not sure they are
> the correct model system to use in this experiment. If we are simply
> looking at binding of myotoxin to lipid then ND would be a go to. However,
> I thought this experiment was more about looking at the change in lipid
> order caused by the interaction with myotoxin. The belt protein causes a
> lateral pressure on the lipid tails that could inhibit changes in the lipid
> order, potentially resulting in us not being able to monitor the changes. I
> would think that liposomes would be a better model system for this
> experiment.
>
> Additionally, I’m not sure that CL would be a good model lipid for
> myotoxin. CL is a lipid that is only found in the mitochondria and the
> testes. Additionally, it has a abnormally high negative charge and lipid
> structure that is not that of a standard lipid. I would think that using
> something more like E. coli total lipids or a muscle lipid mix would be a
> better model system to study.
>
> Best regards,
>
> Harmen Steele, Ph.D.
> Postdoctoral Researcher
> harmen.steele at umontana.edu<mailto:harmen.steele at umontana.edu>
>
> +406 529 9669 - mobile
> Skype Me: harmensteele<callto://harmensteele/>
> Amateur Radio Call Sign: K9HBS
> I am a UM Ally<http://www.umt.edu/umallies/>
>
> This email was sent using 95% recycled binary bits and 99.9% recycled
> electrons.
>
>
> From: Borries Demeler <demeler at gmail.com>
> Date: Thursday, February 20, 2020 at 4:43 PM
> To: "Henrickson, Amy" <amy.henrickson at uleth.ca>
> Cc: "Steele, Harmen" <harmen.steele at umconnect.umt.edu>
> Subject: Re: Samples for NMR
>
> Harmen, we would like to run a CL ND in the NMR as a control for both
> the myotoxin and cytochrome C experiments. We figure the CL would be a good
> model also for myotoxin. Can we use the old samples or should we try a new
> prep without the his tag? We could potentially double-check the quality by
> AUC first. I think Amy also still has some of the 3s material. -Borries
>
> On Thu, Feb 20, 2020, 16:30 Henrickson, Amy <amy.henrickson at uleth.ca
> <mailto:amy.henrickson at uleth.ca>> wrote:
> Hello Harmen,
>
> A while ago you gave me a sample for NMR and it finally looks like we
> may be able to run this.
> However, I wanted to see if you thought that this nanodisk would still
> be stable, it has been stored at 4C since arrival?
>
> I was also wondering what exactly the samples was and what buffer it
> was in and the concentration of the sample?
>
> Thank you,
> Amy Henrickson
>
> ---
> Amy Henrickson B.Sc
> Laboratory Manager Canadian Center for Hydrodynamics Northwest
> Biophysics Consortium Alberta RNA Research and Training Institute
> Department of Chemistry and Biochemistry University of Lethbridge
> 4401 University Drive
> Lethbridge, Alberta, Canada T1K 3M4
> Email: amy.henrickson at uleth.ca<mailto:amy.henrickson at uleth.ca>
> Phone: 403-329-2095
>
>
> ----- End forwarded message -----
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