Improved sensitivity and resolution of in-cell NMR spectra

Abstract

In-cell NMR spectroscopy is a powerful tool to study protein structures and interactions under near physiological conditions in both prokaryotic and eukaryotic living cells. The low sensitivity and resolution of in-cell NMR spectra and limited lifetime of cells over the course of an in-cell experiment have presented major hurdles to wide acceptance of the technique, limiting it to a few select systems. These issues are addressed by introducing the use of the CRINEPT pulse sequence to increase the sensitivity and resolution of in-cell NMR spectra and the use of a bioreactor to maintain cell viability for up to 24h. Application of advanced pulse sequences and bioreactor during in-cell NMR experiments will facilitate the exploration of a wide range of biological processes.

Keywords: Antibiotics; Atomic resolution structure; In vivo biochemistry; In-cell biochemistry; Nucleic acids; Protein interactions; Protein structure; Protein-drug interactions; RNA; Ribosome; Thioredoxin.

Fig. 1.

The in-cell spectra of most folded proteins are undetectable using HSQC NMR spectroscopy. (A). In vitro ¹H{¹⁵N}-HSQC spectrum of Trx. B). ¹H{¹⁵N}-HSQC spectrum of Trx in Escherichia coli. Crosspeaks are not resolved due to an increase in the apparent molecular weight of thioredoxin, only small metabolites are observed.

Fig. 2.

CRINEPT NMR spectroscopy resolves in-cell crosspeaks for proteins with high apparent molecular weights due to quinary interactions. (A) The relative volumes of G52, G66 and G85 crosspeaks are plotted against the CRINEPT transfer delay times. An endogenous tryptophan indole amide peak in the in-cell spectra is used as a reference. (B) ¹H–¹⁵N CRINEPT–HMQC–TROSY spectrum of REDPRO-labeled Trx in E. coli. The G52, G66 and G85 crosspeaks used for optimizing the CRINEPT transfer delay time are indicated.

Fig. 3.

RT in-cell NMR. A gravity siphon drives the continuous flow of fresh medium through the cells.

Fig. 4.

Cell viability is maintained for up to 24 h in a bioreactor. (A) ³¹P NMR spectra showing α-, β- and γ-ATP levels after 6 h for E. coli and HeLa cells in the bioreactor and in E. coli cell lysate. (B) Signal intensity of ³¹P-metabolites over time in E. coli with (squares) and without (circles) M9 flow. (C) Intensity of ³¹P-metabolites over time in HeLa cells with (squares) and without (circles) DMEM flow. (D) Trypan blue staining of cast HeLa cells before in-cell spectroscopy. (E) Trypan blue staining of cast HeLa cells 24 h after casting.

Fig. 5.

Binding of streptomycin to ribosomes changes the quinary structure of Trx in E. coli. Overlay of in-cell ¹H −¹⁵N CRINEPT − HMQC − TROSY spectra of Trx without (red) and with (blue) streptomycin (final spectrum). Residues that broaden in the presence of streptomycin are indicated. Single and double asterisks indicate peaks from metabolites and unassigned side chain protons, respectively. The spectra are shown at the same contour levels. The reference peak used for intensity normalization is indicated by RP.

Fig. 6.

SVD analysis of Trx RT STINT NMR spectra. (A) Distribution of singular values, SV, of each data set index (binding mode) for Trx residues in the presence of streptomycin. (B) The contribution of each residue in response to adding streptomycin for the first (blue) and second (red) binding modes. (C) Surface map of Trx (Protein Data Bank entry 1X0B) showing the quinary interaction surface (red) in the absence of antibiotics. (D) Surface map of Trx showing the quinary interaction surface (red) in the presence of streptomycin. Residues that comprise the interaction surface are indicated.