Method for in-cell NMR?
Pablo Trigo-Mouriño
Thursday, 12 February 2009 18:13 UTC
Hi everyone! I’m a PhD Student at Biomolecular NMR group of USC. Actually I’m working with a integral membrane receptor that is hanging me in trouble, we’re having a lot of problems in the solubilization with detergent procedure so we think that tha use of complete cells that we’re using for expression can be suitable, anyone knows how we can do the stable cellular suspension. Thanks!
Updated 16 February 2009 09:38 UTC
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Replies
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Hi Pablo,
Nature Protocols has published a couple of protocols for in-cell NMR:
In-cell NMR for protein-protein interactions (STINT-NMR) by Burz et al
Unfortunately, these articles require a site license (or pay-per-view) for access.
You may wish to try cross-posting your query to the Nature Protocols Discussion Forum; some of our members might be able to provide further assistance?
Good luck!
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I am a bit more skeptical about this and wonder whether this is a solution state NMR project or whether you plan to do this by solid state? In case of a solution state project, even if you manage to isotope label only your receptor > upon membrane integration the tumbling rate will be very unfavorable and you might (at best) only see the floppy parts of your protein. Remember that ‘membrane’ solution state NMR spectroscopy typically deals with detergent solubilized proteins (a lot of them in SDS micelles, which are reasonably small). Once you integrate a membrane protein into a ‘real’ biological membrane the thing is going to tumble like the whole cell itself.
If you plan to do this by solid state NMR > remember that you will need a high degree of deuteration and complete C13 labeling. In addition, your final protein concentration should be in the mg range. You will need phenomenal expression levels and extremely selective isotope incorporation behavior if this is going to work (which is not very likely).
I would think this over very carefully! you might wanna look at Daron Freedbergs’ publications about on-cell NMR. He is studying natural carbohydrates on the surface of bacteria and his methods work surprisingly well! But those structures are in very different dynamic regimes than integral membrane proteins, so keep that in mind!
good luck!
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I know people use nano-discs for solubilization of membrane proteins. A lot of information is available on the internet about Nano-Discs. It might be a option trying to reconstitute your receptor in them. http://www.nanodiscinc.com/.
I, too think, if your membrane receptor is directed into the membrane when expressed, it would be difficult to gain signal on NMR (due to slower correlation time) as P. Selenko has mentioned already.
Good luck!
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Kielec JM et al Structure. 2009 Mar 11;17(3):345-51
Just came across this article, thought might be helpful for you!
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