Yes, that's a bacterial culture in my NMR tube

There?s linkurl:a pretty slick paper;http://www.nature.com/nmeth/journal/v3/n2/abs/nmeth851.html in the February __Nature Methods__. Alexander Shekhtman, of SUNY-Albany, describes a novel technique called STINT-NMR (for structural interactions using in-cell NMR), which maps a protein?s structural changes in response to protein-protein interactions in vivo. Shekhtman presented his work Tuesday (Jan. 31) at the Keystone Symposium on Structural Genomics, and I got the chance to talk to him about it during this evening?s poster sessions. As Shekhtman explains, most protein-protein mapping strategies (the yeast two-hybrid assay and affinity purification-mass spectrometry, for instance) reveal binary data: either an interaction occurs or it doesn?t. They say nothing about interaction surfaces, nor do they illuminate the broad structural changes that can result from protein docking events. In STINT-NMR Shekhtman sequentially overexpresses two (or more) proteins in bacteria, using different reagents to control the timing of induction. The first protein is grown in isotopically labeled media (that is, media containing N-15), making the newly synthesized protein visible to the NMR. The labeled media is washed away and the second protein is induced, creating a pool of "invisible" proteins. NMR spectra of both the singly and doubly induced cultures are then acquired and compared. If expression of the second protein affects the structure of the first, for instance by a binding event, the result will be changes to the NMR spectra ? that is, chemical shifts will, well, shift. Shekhtman demonstrated the principle using labeled ubiquitin and its interaction partner, STAM2, identifying the interaction interface between the two proteins. Here?s the cool thing: because the bacteria are self-contained, each cell acts as a sort of independent reaction vessel. The culture is thus effectively a collection of tiny NMR tubes. That means the effective protein concentration is much higher than it would be if protein extracts were used, enabling detection of relatively weak interactions. The technique also offers a way to test labile or hard-to-purify proteins. And because it?s in vivo, STINT-NMR avoids the need to purify properly folded proteins. I?d be surprised if STINT-NMR becomes a regular tool for interactome researchers. For one thing, because there's no way to tell if a residue shifts because of direct or indirect effects (eg, by gross conformational changes stemming from distant interactions), the data must be taken with a grain of salt. Nevertheless, the technique could prove valuable to those researchers who want to leverage interaction data in their own research, by providing a relatively easy way to identify potential sites of perturbation, for instance. The only requirements are a known structure, a complete set of resonance assignments, and a technically nimble NMR spectroscopist.

Yes, that's a bacterial culture in my NMR tube

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