[Myotox] Samples for NMR

[Borries Demeler]

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 -----