[Myotox] Samples for NMR

Fri Feb 21 08:33:38 MST 2020

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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