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Abstract

Chemistry

Monitoring Protein-Ligand Interactions in Human Cells by Real-Time Quantitative In-Cell NMR using a High Cell Density Bioreactor

Published: March 9th, 2021

DOI:

10.3791/62323

1Magnetic Resonance Center − CERM, University of Florence, 2Consorzio Interuniversitario Risonanze Magnetiche di Metalloproteine − CIRMMP, 3Neurofarba Department, University of Florence

Abstract

In-cell NMR is a unique approach to observe the structural and dynamic properties of biological macromolecules at atomic resolution directly in living cells. Protein folding, chemical modifications, and conformational changes induced by ligand binding can be observed. Therefore, this method has great potential in the context of drug development. However, the short lifetime of human cells confined in the NMR spectrometer limits the application range of in-cell NMR. To overcome this issue, NMR bioreactors are employed that can greatly improve the cell sample stability over time and, importantly, enable the real-time recording of in-cell NMR spectra. In this way, the evolution of processes such as ligand penetration and binding to the intracellular protein target can be monitored in real time. Bioreactors are often limited by low cell viability at high cell numbers, which results in a trade-off between the overall sensitivity of the experiment and cell viability. We recently reported an NMR bioreactor that maintains a high number of human cells metabolically active for extended periods of time, up to 72 h. This setup was applied to monitor protein-ligand interactions and protein chemical modification. We also introduced a workflow for quantitative analysis of the real-time NMR data, based on multivariate curve resolution. The method provides concentration profiles of the chemical species present in the cells as a function of time, which can be further analyzed to obtain relevant kinetic parameters. Here we provide a detailed description of the NMR bioreactor setup and its application to monitoring protein-ligand interactions in human cells.

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Keywords In cell NMR

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