In-cell NMR applications; what's next?
Philipp Selenko
Thursday, 03 January 2008 12:39 UTC
While in-cell NMR has established itself as a genuine tool for investigating biological macromolecules inside live cells, an open question remains of what will constitute the most powerful in-cell NMR applications in the future? Will it be screening for intracellular(!) ligand interactions (via DNP probably?), will it be analyzing intrinsically unfolded proteins in vivo, will it be drug screening under native conditions, or will it be post-translational protein modification-analyses inside eukaryotic cells? Feel free to post what you think will be the next big thing for in-cell NMR spectroscopy.
Updated 03 April 2008 14:25 UTC
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Personally, I think it is going to be in-cell NMR in mammalian cells (just imagine looking at a brain protein inside brain cells …. daunting isn’t it?)
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I wish I knew what the next big thing for in-cell NMR will be but unfortunately I do not. I would be interested in looking at the influence of kinase activity on transcriptional activity in cell extracts (eg: HeLa or 293T) but I think it is probably still far in the future.
I did want to ask what might be a conceptual roadmap for this field. How will you choose a model system? Are there already good model systems (such as the oocytes, etc.) that will keep you busy for many years before you can apply the ideas to new systems? If I did want to look at kinases and transcription what would be the most basic questions I should think about? For example, presumably the kinase would need to be overexpressed?
Just ideas for now…
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good thinking! But why not rely on endogenous enzymes?
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Fair enough. I remember that you mentioned that on your post in the biomolecular NMR group.
It does give me ideas which I cannot properly articulate just yet. Basically another factor sequesters an active form of a kinase which is essential for transcription. When this inhibition is relieved the active kinase will then be free and transcription can proceed with comcomitant enzymatic activity from both the kinase and RNA polymerase.
There is a small molecule available which apparently functions by breaking up this inactive complex and therefore stimulating transcription. Maybe then in-cell NMR could be used to see the effect on the drug on the complex? I am also trying to think how to study the system without the drug, ie: how to find a unique enzymatic marker that only comes from the transcriptional complex? Perhaps a combination of in-cell NMR and biochemistry to look at aborted transcripts would do it…
So I would find put how to get this very complex system into an NMR tube, record data, take it out of the magnet, run a gel…
Not so easy…but sounds like fun!
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I think they’re all going to be “big things”...but if I could choose which direction to pay attention to, it’d be in-vivo molecular analysis of some kind.
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So, this NMR spectroscopy is all upto eukaryotes?? not prokaryotes??
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