13C direct-detection biomolecular NMR spectroscopy in living cells.

In-cell NMR experiments of labeled biomolecules have been shown to be quite informative about the structure and dynamics of proteins in an environment similar to the natural one. The main approaches have been 1) to overexpress proteins in prokaryotic cells, 8] 2) to inject labeled proteins in oocytes, and 3) to introduce into eukaryotic cells labeled proteins through a target sequence. The most widely used experimental strategy consists in exploiting N isotopic labeling to collect H–N correlation experiments to monitor changes in chemical shift and to map biochemical processes and interactions. H–C correlation experiments are less popular because of the large background signals complicating the analysis of the spectra. Indeed, even not considering the background signals resulting from the labeling of other cellular components besides the protein of interest, the 1% natural abundance of C is sufficient to provide strong signals. This can be partly overcome by correlating C with other heteronuclei, as in the case of H-detected triple resonance experiments that have been used to obtain the sequential assignment of proteins in-cell. 16] In other cases, selective C enrichment has been used, with particular success when focusing on histidines and methyl groups. We have recently pursued the exploitation of C direct detection in solution for biomolecular NMR applications. Thanks to the increase in instrumental sensitivity as well as to improved experimental schemes, C-detected 2D experiments can be acquired in a very short time (a few minutes). Herein, we report the advantages and limits in terms of applications of C-detected experiments acquired on prokaryotic cells containing overexpressed C,N-enriched proteins. In particular, we show that such experiments are quite informative for unfolded parts of proteins for which H–N NMR experiments are less informative. The studied protein is yeast Cox17 (69 amino acids), which is largely unfolded when the seven cysteine residues present in its primary sequence are reduced and largely folded when four of them form interhelix disulfide bonds with a coilhelix-coil-helix fold. Comparative experiments are reported for folded yeast Atx1 (72 amino acids) and for human a-synuclein (140 amino acids), a natively disordered protein which maintains this state also in-cell. The H–N correlation NMR spectrum of Cox17 in Escherichia coli cells provides a fingerprint of the protein incell (Figure 1A). At first sight the spectrum appears to be that of an unfolded protein as the spreading of the H signals is small. This spectrum is close to that of the purified protein in a reducing environment (20 mm dithiothreitol), in which the cysteine residues of the protein are reduced (Figure 1B). However, the poor chemical shift dispersion as well as the increased linewidths typical of in-cell experiments only allow us to resolve a limited number of H–N correlations. The C’–N correlation (CON) spectrum 25] in-cell shows a number of signals (Figure 1C) that is smaller than that of the reduced species in solution (Figure 1D). The C/–C’ correlation (CACO 26] and CBCACO) spectra, which allow us to obtain through simple 2D experiments information on other backbone nuclei (Cs) and on side chains (Cs),