Abstract

Protein–protein interactions, PPIs, underlie most cellular processes, but many PPIs depend on a particular metabolic state that can only be observed in live, actively metabolizing cells. Real time in-cell NMR spectroscopy, RT-NMR, utilizes a bioreactor to maintain cells in an active metabolic state. Improvement in bioreactor technology maintains ATP levels at >95% for up to 24 hours, enabling protein overexpression and a previously undetected interaction between prokaryotic ubiquitin-like protein, Pup, and mycobacterial proteasomal ATPase, Mpa, to be detected. Singular value decomposition, SVD, of the NMR spectra collected over the course of Mpa overexpression easily identified the PPIs despite the large variation in background signals due to the highly active metabolome.

Highlights

  • Protein–protein interactions, PPIs, underlie most cellular processes, but many PPIs depend on a particular metabolic state that can only be observed in live, actively metabolizing cells

  • During this time waste is being produced, and the pH and redox state of the cell is changing, which can lead to changes in the NMR spectra as the protein reacts to the cellular environment[12]

  • Changes in the cellular environment lead to cell death and leakage of target protein from the cell resulting in NMR spectra that are a combination of in-cell and free protein

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Summary

Introduction

Protein–protein interactions, PPIs, underlie most cellular processes, but many PPIs depend on a particular metabolic state that can only be observed in live, actively metabolizing cells. Improvement in bioreactor technology maintains ATP levels at >95% for up to 24 hours, enabling protein overexpression and a previously undetected interaction between prokaryotic ubiquitin-like protein, Pup, and mycobacterial proteasomal ATPase, Mpa, to be detected. The original design of our bioreactor sustained metabolic energy levels in E.coli for up to 24 h with ~40% loss of ATP, ADP, NAD+, and NAD(H) concentrations, consistent with >90% cell viability and limited cell growth[13]. By maintaining a highly active cellular metabolism ATP-dependent functional PPIs can be identified by combining real time, RT, and structural interactions, STINT8, in-cell NMR13. The Pup-proteasome system is implicated in persistent infections of macrophages by Mtb and is recognized as an important drug target[23,24,25]

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