In-Cell NMR Spectroscopy: Inhibition of Autologous Protein Expression Reduces Escherichia coli Lysis

Abstract

Structural studies by in-cell nuclear magnetic resonance are a developing new field of research, and their objective is to obtain structural information of proteins and other biological macromolecules in the cytoplasm of Escherichia coli cells. The major limitation of in-cell experiments is cell lysis that occurs during the experiments. In this article, we describe how inhibition of autologous expression by rifampicin at a high concentration decreases cell lysis in E. coli. We suggest that rifampicin is acting in the programmed cell death gene system MazEF, which is triggered by stress conditions and ultimately leads to cell lysis.

In-Cell Biochemistry Using NMR Spectroscopy

“…To rule out the possibility that the NMR spectrum of Ubiquitin is due to extracellular protein [16], [17], after obtaining the in-cell NMR spectrum, the cells were centrifuged and the supernatant was examined. No NMR spectrum of Ubiquitin was observed above noise level (Fig.…”

“…All NMR experiments were performed using a Bruker Avance 700 MHz NMR spectrometer equipped with a cryoprobe. The cryoprobe affords a four-fold increase in sensitivity allowing data collection within ∼1 hr for an individual experiment; this is critical to minimize cell leakage [17]. We used a watergate version of the 1 H{ 15 N}-HSQC spectrum.…”

In-cell NMR spectroscopy

“…This shortfall can be compensated for by increasing the intracellular concentration of the target, by increasing the number of scans per experiment and by using a ultrasensitive cryoprobe. Stabilizing the cells by using known cell protectants, such as glycerol or sucrose [37], or alginate encapsulation [38], may also extend the in-cell NMR acquisition time. Finally, the sensitivity of the resulting in-cell spectrum may be increased by using transverse optimized spectroscopy (TROSY-HSQC) for high molecular weight complexes that have slow tumbling times.…”

“…Another important factor that limits the acquisition time of in-cell NMR experiments is cell lysis, which results in leakage of labeled target from the cells [37]. Xie et al .…”

CHAPTER 4. In‐Cell NMR Spectroscopy to Study Protein–Drug Interactions

“…8 Another important factor that limits the acquisition time of in-cell NMR experiments is cell lysis, which also results in leakage of labeled target from the cells. 9 Stabilizing cells by using known cell protectants, such as glycerol or sucrose, 9 may also extend the in-cell NMR acquisition time. Control experiments are performed to ensure that the NMR signal arises from proteins located inside the cell.…”