Going SAILing anytime soon?
Michael Durney
Wednesday, 26 November 2008 15:42 UTC
Advances in NMR have now made it possible to study the dynamics and structures of very large systems using several different selective labeling methods. Proteins can be labeled uniformly with carbon, nitrogen and deuterium and selectively by amino acid type, by simple expression in bacteria. Simple in vitro modifications can be used to paramagnetically label residues to gain access to long-range information. Segmental labeling by domain is another long-promised area that is finally becoming more generally applicable. However the potentially most powerful labeling method for high-resolution structure determination is still considered by many to be technically very challenging, namely stereo-array isotope labeling, known as SAIL. While this technique has been shown to open the road to automated structure determination of large systems routine application is still not a reality due to the high cost of the SAIL amino acids and the required optimization of cell-free protein expression. Luckily centers such as the Center for Eukaryotic Structural Genomics at UW Madison offer expertise and training in this technology.
Are many peope interested in this technique? Has anyone tried cell-free expression in their lab? Do you have a dream project that you would like to apply this technology too? As NMR spectroscopists we are very lucky to have such useful NMR-active isotopes but the size limitation can hold some projects back. If someone says “that is too big, NMR can’t do it.” I think we should say “Yes, we can!”. If it’s worth it of course!
Any thoughts would be appreciated….
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Replies
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I don’t know much about SAIL, but I think it definitely belongs in the canon of isotope labeling options the biomolecular NMR spectroscopists should consider. The first thing to ask though is, what do you hope to accomplish? If you’re in the business of solving a several hundred kDa protein structure/complex from scratch, I’d say you’re asking the wrong questions. Now, if you’re interested in dynamics or ligand binding or similar questions, there’s a great deal you can do. Proteins of that size suffer tremendously from H-H dipolar interactions and you have to chose your battles carefully. If SAIL offers that sort of flexibility, then I would definitely be interested in using it one day.
That being said, a LOT more needs to be done on this front for large nucleic acids. There are some options for specific labeling, but not many commercially available. The manageable size for RNAs and DNAs is only in the few 10s of kDa (<100 nts), and that may be pushing it!
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Thanks for your reply,
An appreciable reduction is relaxation rates for larger proteins is one of the benefits of SAIL. Also, NOE spectra are simplified considerably. I don’t think either that several hundred kDa structures are possible or necessary but perhaps the range 50-100 kDa can be explored in more detail. And yes, dynamics and interactions of large assemblies has become accessible even before SAIL.
For nucleic acids and RNA in particular I think that combinations of nucleotide-specific labeling, deuteration, and segmental ligation will bring us structural data on biologically-relevant RNAs with tertiary interactions. The area of rNTPs labeled at specific positions is also something to watch maybe. Compared to protein expression in cells or extracts the in vitro RNA transcription system definitely has advantages due to it’s simplicity. Let’s see what the future brings!
Has anyone tried cell-free expression for protein NMR samples? If so, what are you experiences with it?
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