In-cell NMR as a sensitive tool to monitor physiological condition of Escherichia coli

The in-cell NMR technique offers significant insights into the structure and function of heterologous proteins in the physiological intracellular environment at an atomic resolution. Escherichia coli (E. coli) is one of the most widely used host cells for heterologous protein expression in structural biological studies as well as for in-cell NMR studies to investigate fundamental structural characteristics and the physiochemistry of certain proteins and their intermolecular interactions under physiological conditions. However, in many cases, it is not easy to obtain well-resolved in-cell NMR spectra because the detectability and resolution of these spectra are significantly influenced by intracellular factors such as nonspecific intermolecular interactions. In this study, we re-examined the experimental parameters of E. coli in-cell NMR and found that the detectability and resolution of the NMR spectra clearly depended on the growth phase of the host cells. Furthermore, the detectability and resolution of the E. coli in-cell NMR spectra correlated with the soluble fraction amounts of the expressed target protein. These results indicate that the E. coli in-cell NMR spectrum of a target protein is a useful tool for monitoring the intracellular conditions of the host cell and for establishing the appropriate cultivation conditions for protein overexpression.


E. coli in-cell NMR tips demonstrated in this study
When higher-quality E. coli in-cell NMR spectra was measured, the following phenomena were seen prior to the start of the in-cell NMR measurements in many cases: (1) The intensity of the 2 H lock signal of 2 H2O was sufficiently high, indicating that locking of the magnetic field can be accomplished smoothly. Conversely, when the intensity of the 2 H lock signal of 2 H2O was weak, the locking was prone to being aborted and the quality of the in-cell NMR spectra was low even if locking of the static magnetic field was forced to work by increasing 2 H lock power. Serber and colleagues have reported that too great a cell density in an NMR sample tube stalled the sensitivity of the 2 H lock signal and led to a loss of sensitivity of shimming (Serber et al. 2001a, b;Reckel et al. 2007). In addition to this insight of Serber's, our results indicated that attenuation of the 2 H lock signal is a general phenomenon seen not only when there is too great a cell density in the NMR sample tube but also when the quality of the in-cell NMR spectra is low.
(2) Automatic shimming programs (such as TopShim, provided by Bruker (Fällanden, Switzerland)) work successfully without being aborted, and the standard deviation of the final B0 value (which is an indicator of the homogeneity of the static magnetic field through the sample, measured by TopShim) after running the automatic shimming could be less than 0.5 Hz. When the automatic shimming program was aborted or the standard deviation of the final B0 value after automatic shimming was not improved above around 1-2 Hz, it was hardly worth continuing to set up the in-cell NMR measurement of the current sample. The abortion of, or poor results from, 3 automatic shimming has been attributed to the weak intensity of the 2 H lock signal, as described above.
The assignment of E. coli in-cell NMR-specific unknown signals and the evaluation of their origin molecules.
We attempted to identify the origin of identified signals because assignment of these signals may help not only to elucidate details of the biological nature of E. coli cell growth but also to develop the E. coli in-cell NMR methodology, enabling the elimination or filtering of undesired signals. Overexpression of the GB1 protein was started by adding isopropyl β-D-1-thiogalactopyranoside (IPTG) at OD600 = 1.80 to appear the unknown signals, and its 3D TROSY-HNCACB spectrum was measured (Supplemental Fig. S13). Its spectrum indicated that two of the unknown signals were derived from glycine residues (Supplemental Fig. S13A). However, although correlation signals between inter-residual Cα(i-1) and Cα(i) of glycine-candidate appeared in the in-cell 3D TROSY-HNCACB spectrum (Supplemental Fig. S13A), the origin of the inter-residue (i-1) correlation signal could not be assigned due to the corresponding signals being missing.
This assumption was further supported from the results of the glycine-selective inverse isotope labeling experiments and glycine-selective 1 H-15 N HSQC measurements using the MUSIC pulse scheme (G-HSQC) (Schubert et al. 1999;Schubert et al. 2001) (Supplemental Figs. S13B and S13C). In the glycine-selectively 14 N-labeled sample, the signal intensity of those unknown signals decreased significantly compared to that of a uniformly 15 N-labeled sample (Supplemental Fig. S13B). Furthermore, the G-HSQC spectrum also provided support that the unknown first signal was certainly glycine (Supplemental Fig. S13C). The second of the two glycine-candidate signals (unknown signal No. 2) could not be observed in the G-HSQC due to weak signal intensity. From these results, it was assumed that the appearance of several unknown and undesired in-cell NMR signals could be suppressed as far as possible by glycine-selective isotopic unlabeling or coherence filtering.
It is known that D-alanine in a peptidoglycan of E. coli (denoted as red circles on the left panel of Supplemental Fig. S13D) can be replaced by glycine (Vollmer et al. 2008). In addition, the chemical shift of the inter-residual Cα(i-1) correlation signals of the HNCACB of the candidate of glycine was close to the Cα of m-A2pm, a component of the cross-linking region between the glycan chains in a peptidoglycan of the E. coli; it was in the (i-1) position of the D-alanine (Supplemental Fig. S13D). These results suggest that the glycine-candidate unknown signals were derived from peptide fragments containing m-A2pm-Gly bis-amino acid, generated from peptidoglycan of the E. coli host cells in a late phase of the cell growth.
In this study, we demonstrated the possibility that unknown NMR signals appearing uniquely in the E. coli in-cell NMR measurements were derived from peptidoglycan fragments containing glycine residue. However, in order to make definite identifications of the origin molecules in the future, further experiments using other techniques such as solid-state NMR are needed as there are many difficulties and limitations to the solution NMR approach in analyzing peptidoglycan, a large molecular assembly, even when fragmented. The NMR spectra of the lysate were measured after cell debris was eliminated by centrifugation at 12,000 g for 10 min at 4 °C (Xu et al. 2014). The OD600 value at the timing of the addition of IPTG is indicated at the top left corner of each NMR spectra.