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A thermosensitive gel matrix for bioreactor-assisted in-cell NMR of nucleic acids and proteins
Sep 10, 2023 - 2:39 AM - by nmrlearner
nmrlearner's Avatar A thermosensitive gel matrix for bioreactor-assisted in-cell NMR of nucleic acids and proteins

Abstract

Introducing the flow through the bioreactor has revolutionized in-cell NMR spectroscopy by prolonging the measurement time available to acquire spectral information about biomacromolecules in metabolically active cells. Bioreactor technology relies on immobilizer matrices, which secure cells in the active volume of the NMR coil and enable uniform perfusion of the growth medium, supplying fresh nutrients to the cells while removing toxic byproducts of their metabolism. The main drawbacks of commonly used matrices include the inability to recover intact cells post-measurement for additional analyses and/or requirements for specific operating temperatures. Here, we report on the development and characterization of a set of thermosensitive and nontoxic triblock copolymers based on poly(D,L-lactide)-b-poly(ethylene glycol)-b-poly(D,L-lactide) (PLA-PEG-PLA). Here, we show for the first time that these copolymers are suitable as immobilizer matrices for the acquisition of in-cell NMR spectra of nucleic acids and proteins over a commonly used sample temperature range of 15â??40Â*°C and, importantly, allow recovery of cells after completion of in-cell NMR spectra acquisition. We compared the performances of currently used matrices in terms of cell viability (dye exclusion assays), cellular metabolism (1D 31P NMR), and quality of in-cell NMR spectra of two model biomacromolecules (hybrid double-stranded/i-motif DNA and ubiquitin). Our results demonstrate the suitability and advantages of PLA-PEG-PLA copolymers for application in bioreactor-assisted in-cell NMR.



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Postdoctoral Scholar – Structural biology, NMR, protein chemistry ... - American Chemical Society
Sep 10, 2023 - 2:39 AM - by nmrlearner
nmrlearner's Avatar Postdoctoral Scholar – Structural biology, NMR, protein chemistry ... - American Chemical Society

Postdoctoral Scholar – Structural biology, NMR, protein chemistry ... American Chemical Society Read here
0 Replies | 209 Views
[NMR paper] A thermosensitive gel matrix for bioreactor-assisted in-cell NMR of nucleic acids and proteins
Sep 10, 2023 - 2:39 AM - by nmrlearner
nmrlearner's Avatar A thermosensitive gel matrix for bioreactor-assisted in-cell NMR of nucleic acids and proteins

Introducing the flow through the bioreactor has revolutionized in-cell NMR spectroscopy by prolonging the measurement time available to acquire spectral information about biomacromolecules in metabolically active cells. Bioreactor technology relies on immobilizer matrices, which secure cells in the active volume of the NMR coil and enable uniform perfusion of the growth medium, supplying fresh nutrients to the cells while removing toxic byproducts of their metabolism. The main drawbacks of...

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0 Replies | 162 Views
[NMR paper] Three Decades of REDOR in Protein Science: A Solid-State NMR Technique for Distance Measurement and Spectral Editing
Sep 10, 2023 - 2:39 AM - by nmrlearner
nmrlearner's Avatar Three Decades of REDOR in Protein Science: A Solid-State NMR Technique for Distance Measurement and Spectral Editing

Solid-state NMR (ss-NMR) is a powerful tool to investigate noncrystallizable, poorly soluble molecular systems, such as membrane proteins, amyloids, and cell walls, in environments that closely resemble their physical sites of action. Rotational-echo double resonance (REDOR) is an ss-NMR methodology, which by reintroducing heteronuclear dipolar coupling under magic angle spinning conditions provides intramolecular and intermolecular distance restraints at the atomic level. In addition, REDOR can...

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0 Replies | 272 Views
[NMR paper] Improving Geometric Validation Metrics and Ensuring Consistency with Experimental Data through TrioSA: An NMR Refinement Protocol
Sep 10, 2023 - 2:39 AM - by nmrlearner
nmrlearner's Avatar Improving Geometric Validation Metrics and Ensuring Consistency with Experimental Data through TrioSA: An NMR Refinement Protocol

Protein model refinement a the crucial step in improving the quality of a predicted protein model. This study presents an NMR refinement protocol called TrioSA (torsion-angle and implicit-solvation-optimized simulated annealing) that improves the accuracy of backbone/side-chain conformations and the overall structural quality of proteins. TrioSA was applied to a subset of 3752 solution NMR protein structures accompanied by experimental NMR data: distance and dihedral angle restraints. We...

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0 Replies | 206 Views
HIV-1 Vpu protein forms stable oligomers in aqueous solution via its ... - Nature.com
Sep 08, 2023 - 8:28 PM - by nmrlearner
nmrlearner's Avatar HIV-1 Vpu protein forms stable oligomers in aqueous solution via its ... - Nature.com

HIV-1 Vpu protein forms stable oligomers in aqueous solution via its ... Nature.com Read here
0 Replies | 228 Views
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