Combined Rapid Injection NMR and Simulation Approach to Probe Redox-Dependent Pathway Control in Living Cells

Research output: Contribution to journalJournal articleResearchpeer-review

Abstract

Dynamic response of intracellular reaction cascades to changing environments is a hallmark of living systems. As metabolism is complex, mechanistic models have gained popularity for describing the dynamic response of cellular metabolism and for identifying target genes for engineering. At the same time, the detailed tracking of transient metabolism in living cells on the sub-minute timescale has become amenable using dynamic nuclear polarization enhanced 13C NMR. Here, we suggest an approach combining in-cell NMR spectroscopy with perturbation experiments and modeling to obtain evidence that the bottlenecks of yeast glycolysis depend on intracellular redox state. In pre-steady state glycolysis, pathway bottlenecks shift from downstream to upstream reactions within few seconds, consistent with a rapid decline in the NAD+/NADH ratio. Simulations using mechanistic models reproduce the experimentally observed response and help identify unforeseen biochemical events. Remaining inaccuracies in the computational models can be identified experimentally. The combined use of rapid injection NMR spectroscopy and in silico simulations provides a promising method for characterizing cellular reactions with increasing mechanistic detail.
Original languageEnglish
JournalAnalytical Chemistry
Volume91
Issue number8
Pages (from-to)5395-5402
Number of pages8
ISSN0003-2700
DOIs
Publication statusPublished - 2019

Keywords

  • Biocatalysis
  • In vivo spectroscopy
  • Metabolism
  • NMR spectroscopy
  • Simulation

Cite this

@article{44e979e1bb8f4a97b911c11b623bed5e,
title = "Combined Rapid Injection NMR and Simulation Approach to Probe Redox-Dependent Pathway Control in Living Cells",
abstract = "Dynamic response of intracellular reaction cascades to changing environments is a hallmark of living systems. As metabolism is complex, mechanistic models have gained popularity for describing the dynamic response of cellular metabolism and for identifying target genes for engineering. At the same time, the detailed tracking of transient metabolism in living cells on the sub-minute timescale has become amenable using dynamic nuclear polarization enhanced 13C NMR. Here, we suggest an approach combining in-cell NMR spectroscopy with perturbation experiments and modeling to obtain evidence that the bottlenecks of yeast glycolysis depend on intracellular redox state. In pre-steady state glycolysis, pathway bottlenecks shift from downstream to upstream reactions within few seconds, consistent with a rapid decline in the NAD+/NADH ratio. Simulations using mechanistic models reproduce the experimentally observed response and help identify unforeseen biochemical events. Remaining inaccuracies in the computational models can be identified experimentally. The combined use of rapid injection NMR spectroscopy and in silico simulations provides a promising method for characterizing cellular reactions with increasing mechanistic detail.",
keywords = "Biocatalysis, In vivo spectroscopy, Metabolism, NMR spectroscopy, Simulation",
author = "Jensen, {Pernille Rose} and Marta Matos and Nikolaus Sonnenschein and Sebastian Meier",
year = "2019",
doi = "10.1021/acs.analchem.9b00660",
language = "English",
volume = "91",
pages = "5395--5402",
journal = "Analytical Chemistry",
issn = "0003-2700",
publisher = "American Chemical Society",
number = "8",

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TY - JOUR

T1 - Combined Rapid Injection NMR and Simulation Approach to Probe Redox-Dependent Pathway Control in Living Cells

AU - Jensen, Pernille Rose

AU - Matos, Marta

AU - Sonnenschein, Nikolaus

AU - Meier, Sebastian

PY - 2019

Y1 - 2019

N2 - Dynamic response of intracellular reaction cascades to changing environments is a hallmark of living systems. As metabolism is complex, mechanistic models have gained popularity for describing the dynamic response of cellular metabolism and for identifying target genes for engineering. At the same time, the detailed tracking of transient metabolism in living cells on the sub-minute timescale has become amenable using dynamic nuclear polarization enhanced 13C NMR. Here, we suggest an approach combining in-cell NMR spectroscopy with perturbation experiments and modeling to obtain evidence that the bottlenecks of yeast glycolysis depend on intracellular redox state. In pre-steady state glycolysis, pathway bottlenecks shift from downstream to upstream reactions within few seconds, consistent with a rapid decline in the NAD+/NADH ratio. Simulations using mechanistic models reproduce the experimentally observed response and help identify unforeseen biochemical events. Remaining inaccuracies in the computational models can be identified experimentally. The combined use of rapid injection NMR spectroscopy and in silico simulations provides a promising method for characterizing cellular reactions with increasing mechanistic detail.

AB - Dynamic response of intracellular reaction cascades to changing environments is a hallmark of living systems. As metabolism is complex, mechanistic models have gained popularity for describing the dynamic response of cellular metabolism and for identifying target genes for engineering. At the same time, the detailed tracking of transient metabolism in living cells on the sub-minute timescale has become amenable using dynamic nuclear polarization enhanced 13C NMR. Here, we suggest an approach combining in-cell NMR spectroscopy with perturbation experiments and modeling to obtain evidence that the bottlenecks of yeast glycolysis depend on intracellular redox state. In pre-steady state glycolysis, pathway bottlenecks shift from downstream to upstream reactions within few seconds, consistent with a rapid decline in the NAD+/NADH ratio. Simulations using mechanistic models reproduce the experimentally observed response and help identify unforeseen biochemical events. Remaining inaccuracies in the computational models can be identified experimentally. The combined use of rapid injection NMR spectroscopy and in silico simulations provides a promising method for characterizing cellular reactions with increasing mechanistic detail.

KW - Biocatalysis

KW - In vivo spectroscopy

KW - Metabolism

KW - NMR spectroscopy

KW - Simulation

U2 - 10.1021/acs.analchem.9b00660

DO - 10.1021/acs.analchem.9b00660

M3 - Journal article

C2 - 30896922

VL - 91

SP - 5395

EP - 5402

JO - Analytical Chemistry

JF - Analytical Chemistry

SN - 0003-2700

IS - 8

ER -