Combined In-Cell NMR and Simulation Approach to Probe Redox-Dependent Pathway Control

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 13 C 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.

Pancreatic β-cells respond to fuel pressure with an early metabolic switch

“…S5 ). These reactions have previously been shown to respond to redox changes in yeast and mammalian cancer cells 20 , 21 . Since these metabolic pathways are part of the strongly conserved central carbon metabolism we suggest that a similar mechanism might be used as defense mechanism in β-cells, as well.…”

In-cell NMR: Why and how?

“…Dissolution-DNP experiments have also been used to study microorganism metabolism. Researchers affiliated to the Carlsberg Laboratory directed their investigations towards yeast glycolysis and CO 2 /ethanol production: upon feeding S. cerevisiae cell suspensions with hyperpolarized (and deuterated) 13 C-glucose or fructose, they observed the successive appearance of a dozen intermediate products from central carbon metabolism [1116], its inhibition by sulfite notably via pyruvate-sulfite adducts formation [1117], acetaldehyde accumulation upon acetate exposure [1118] and its impact on the intracellular redox status of the glycolysis pathway [1119]. They also used hyperpolarized 13 Cacetate to monitor its influx: because the acetate chemical shift depends on pH, two peaks corresponding to extracellular (low pH) and intracellular (pH$7) acetate could be observed by NMR in real-time [1118].…”

Esterase Activity of Serum Albumin Studied by 1H NMR Spectroscopy and Molecular Modelling

“…That is why such a delay is applicable for measurement of kinetics of albumin true esterase activity, however, to study the reaction of Sudlow site II (which occurs during the first 5 min of BSA-NPA interaction), faster NMR methods should be applied [ 16 , 17 ]. For example, rapid injection NMR spectroscopy allow the observation of fast chemical reactions and unstable intermediates [ 18 , 19 ].…”