Related ArticlesReal-time quantitative in-cell NMR: ligand binding and protein oxidation monitored in human cells using multivariate curve resolution.
Anal Chem. 2020 Jun 18;:
Authors: Luchinat E, Barbieri L, Campbell TF, Banci L
Abstract
In-cell NMR can investigate at atomic resolution protein conformational changes, such as those induced by drug binding or chemical modifications, directly in living human cells, showing great potential in the context of drug development as it can provide an early assessment of drug potency. NMR bioreactors can greatly improve the cell sample stability over time and, more importantly, allow recording in-cell NMR data in real time to monitor the evolution of intracellular processes, thus providing unique insights in the kinetics of drug-target interactions. However, current implementations are limited by low cell viability at >24 hours times, the reduced sensitivity compared to 'static' experiments and the lack of protocols for automated and quantitative analysis of large amounts of data. Here, we report an improved bioreactor design, which maintains human cells alive and metabolically active for up to 72 hours, and a semi-automated workflow for quantitative analysis of real-time in-cell NMR data relying on Multivariate Curve Resolution (MCR). We apply this setup to monitor protein-ligand interactions and protein oxidation in real time. High-quality concentration profiles can be obtained from noisy 1D and 2D NMR data with high temporal resolution, allowing further analysis by fitting with kinetic models. This unique approach can therefore be applied to investigate complex kinetic behaviours of macromolecules in a cellular setting, and could be extended in principle to any real-time NMR application in live cells.
PMID: 32551584 [PubMed - as supplied by publisher]
[NMR paper] Multiplexed real-time NMR GTPase assay for simultaneous monitoring of multiple GEF activities from human cancer cells and organoids.
Multiplexed real-time NMR GTPase assay for simultaneous monitoring of multiple GEF activities from human cancer cells and organoids.
Multiplexed real-time NMR GTPase assay for simultaneous monitoring of multiple GEF activities from human cancer cells and organoids.
J Am Chem Soc. 2018 Mar 15;:
Authors: Gebregiworgis T, Marshall CB, Nishikawa T, Radulovich N, Sandi MJ, Fang Z, Rottapel R, Tsao MS, Ikura M
Abstract
Small GTPases (sGTPases) are critical switch-like regulators that mediate several important cellular functions and...
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03-16-2018 12:12 PM
[NMR paper] Salt-mediated oligomerization of the mouse prion protein monitored by real time NMR.
Salt-mediated oligomerization of the mouse prion protein monitored by real time NMR.
Related Articles Salt-mediated oligomerization of the mouse prion protein monitored by real time NMR.
J Mol Biol. 2017 May 11;:
Authors: Sengupta I, Bhate SH, Das R, Udgaonkar JB
Abstract
The prion protein forms ß-rich soluble oligomers in vitro at pH4 in the presence of physiological concentrations of salt. In the absence of salt, oligomerization and misfolding does not take place in an experimentally tractable timescale. While it is well...
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05-16-2017 10:27 PM
Salt-mediated oligomerization of the mouse prion protein monitored by real time NMR
Salt-mediated oligomerization of the mouse prion protein monitored by real time NMR
Publication date: Available online 11 May 2017
Source:Journal of Molecular Biology</br>
Author(s): Ishita Sengupta, Suhas H. Bhate, Ranabir Das, Jayant B. Udgaonkar</br>
The prion protein forms ß-rich soluble oligomers in vitro at pH4 in the presence of physiological concentrations of salt. In the absence of salt, oligomerization and misfolding does not take place in an experimentally tractable timescale. While it is well established that a lowering of pH facilitates...
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05-11-2017 12:28 PM
[NMR paper] Glycosaminoglycan Binding and Non-Endocytic Membrane Translocation of Cell-Permeable Octaarginine Monitored by Real-Time In-Cell NMR Spectroscopy.
Glycosaminoglycan Binding and Non-Endocytic Membrane Translocation of Cell-Permeable Octaarginine Monitored by Real-Time In-Cell NMR Spectroscopy.
Related Articles Glycosaminoglycan Binding and Non-Endocytic Membrane Translocation of Cell-Permeable Octaarginine Monitored by Real-Time In-Cell NMR Spectroscopy.
Pharmaceuticals (Basel). 2017 Apr 15;10(2):
Authors: Takechi-Haraya Y, Aki K, Tohyama Y, Harano Y, Kawakami T, Saito H, Okamura E
Abstract
Glycosaminoglycans (GAGs), which are covalently-linked membrane proteins at the cell...
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04-20-2017 06:14 PM
[NMR paper] Real-time kinetics of high-mobility group box 1 oxidation in extracellular fluids studied by in situ protein NMR spectroscopy.
Real-time kinetics of high-mobility group box 1 oxidation in extracellular fluids studied by in situ protein NMR spectroscopy.
Real-time kinetics of high-mobility group box 1 oxidation in extracellular fluids studied by in situ protein NMR spectroscopy.
J Biol Chem. 2013 Feb 27;
Authors: Zandarashvili L, Sahu D, Lee K, Lee YS, Singh P, Rajarathnam K, Iwahara J
Abstract
Some extracellular proteins are initially secreted in the reduced form via a non-canonical pathway bypassing the endoplasmic reticulum, and become oxidized in the...
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03-01-2013 09:57 PM
Kinetic analysis of protein aggregation monitored by real-time 2D solid-state NMR spectroscopy
Kinetic analysis of protein aggregation monitored by real-time 2D solid-state NMR spectroscopy
Abstract It is shown that real-time 2D solid-state NMR can be used to obtain kinetic and structural information about the process of protein aggregation. In addition to the incorporation of kinetic information involving intermediate states, this approach can offer atom-specific resolution for all detectable species. The analysis was carried out using experimental data obtained during aggregation of the 10.4 kDa Crh protein, which has been shown to involve a partially unfolded intermediate...
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01-27-2011 04:31 AM
Kinetic analysis of protein aggregation monitored by real-time 2D solid-state NMR spectroscopy.
Kinetic analysis of protein aggregation monitored by real-time 2D solid-state NMR spectroscopy.
Kinetic analysis of protein aggregation monitored by real-time 2D solid-state NMR spectroscopy.
J Biomol NMR. 2011 Jan 21;
Authors: Etzkorn M, Böckmann A, Baldus M
It is shown that real-time 2D solid-state NMR can be used to obtain kinetic and structural information about the process of protein aggregation. In addition to the incorporation of kinetic information involving intermediate states, this approach can offer atom-specific resolution for all...
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01-22-2011 01:52 PM
[NMR paper] Folding of a beta-sheet protein monitored by real-time NMR spectroscopy.
Folding of a beta-sheet protein monitored by real-time NMR spectroscopy.
Related Articles Folding of a beta-sheet protein monitored by real-time NMR spectroscopy.
J Mol Biol. 2003 May 16;328(5):1161-71
Authors: Mizuguchi M, Kroon GJ, Wright PE, Dyson HJ
At low ionic strength, apoplastocyanin forms an unfolded state under non-denaturing conditions. The refolding of this state is sufficiently slow to allow real-time NMR experiments to be performed. Folding of apoplastocyanin, initiated by the addition of salt and followed by real-time 2D 1H-15N...
Real-time quantitative in-cell NMR: ligand binding and protein oxidation monitored in human cells using multivariate curve resolution.
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