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DNP-assisted solid-state NMR enables detection of proteins at nanomolar concentrations in fully protonated cellular milieu
Mar 24, 2024 - 6:32 AM - by nmrlearner
nmrlearner's Avatar DNP-assisted solid-state NMR enables detection of proteins at nanomolar concentrations in fully protonated cellular milieu

Abstract

With the sensitivity enhancements conferred by dynamic nuclear polarization (DNP), magic angle spinning (MAS) solid state NMR spectroscopy experiments can attain the necessary sensitivity to detect very low concentrations of proteins. This potentially enables structural investigations of proteins at their endogenous levels in their biological contexts where their native stoichiometries with potential interactors is maintained. Yet, even with DNP, experiments are still sensitivity limited. Moreover, when an isotopically-enriched target protein is present at physiological levels, which typically range from low micromolar to nanomolar concentrations, the isotope content from the natural abundance isotopes in the cellular milieu can outnumber the isotope content of the target protein. Using isotopically enriched yeast prion protein, Sup35NM, diluted into natural abundance yeast lysates, we optimized sample composition. We found that modest cryoprotectant concentrations and fully protonated environments support efficient DNP. We experimentally validated theoretical calculations of the limit of specificity for an isotopically enriched protein in natural abundance cellular milieu. We establish that, using pulse sequences that are selective for adjacent NMR-active nuclei, proteins can be specifically detected in cellular milieu at concentrations in the hundreds of nanomolar. Finally, we find that maintaining native stoichiometries of the protein of interest to the components of the cellular environment may be important for proteins that make specific interactions with cellular... [Read More]
0 Replies | 138 Views
Decorating phenylalanine side-chains with triple labeled 13C/19F/2H isotope patterns
Mar 24, 2024 - 6:32 AM - by nmrlearner
nmrlearner's Avatar Decorating phenylalanine side-chains with triple labeled 13C/19F/2H isotope patterns

Abstract

We present an economic and straightforward method to introduce 13C-19F spin systems into the deuterated aromatic side chains of phenylalanine as reporters for various protein NMR applications. The method is based on the synthesis of [4-13C, 2,3,5,6-2H4] 4-fluorophenylalanine from the commercially available isotope sources [2-13C] acetone and deuterium oxide. This compound is readily metabolized by standard Escherichia coli overexpression in a glyphosate-containing minimal medium, which results in high incorporation rates in the corresponding target proteins.



Source: Journal of Biomolecular NMR
0 Replies | 43 Views
Quantifying acute-phase inflammation proteins by nuclear magnetic resonance spectrometry - Medical Xpress
Mar 24, 2024 - 6:32 AM - by nmrlearner
nmrlearner's Avatar Quantifying acute-phase inflammation proteins by nuclear magnetic resonance spectrometry Medical Xpress
Quantifying acute-phase inflammation proteins by nuclear magnetic resonance spectrometry - Medical Xpress
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0 Replies | 64 Views
Nuclear magnetic resonance scanner for individual proteins - mpg.de
Mar 24, 2024 - 6:32 AM - by nmrlearner
nmrlearner's Avatar Nuclear magnetic resonance scanner for individual proteins mpg.de
Nuclear magnetic resonance scanner for individual proteins - mpg.de
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0 Replies | 53 Views
[NMR paper] Unraveling motion in proteins by combining NMR relaxometry and molecular dynamics simulations: A case study on ubiquitin
Mar 11, 2024 - 10:30 PM - by nmrlearner
nmrlearner's Avatar Unraveling motion in proteins by combining NMR relaxometry and molecular dynamics simulations: A case study on ubiquitin

Nuclear magnetic resonance (NMR) relaxation experiments shine light onto the dynamics of molecular systems in the picosecond to millisecond timescales. As these methods cannot provide an atomically resolved view of the motion of atoms, functional groups, or domains giving rise to such signals, relaxation techniques have been combined with molecular dynamics (MD) simulations to obtain mechanistic descriptions and gain insights into the functional role of side chain or domain motion. In this work,...

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0 Replies | 64 Views
[NMR paper] Sedimentation of large, soluble proteins up to 140 kDa for 1H-detected MAS NMR and 13C DNP NMR - practical aspects
Mar 11, 2024 - 10:30 PM - by nmrlearner
nmrlearner's Avatar Sedimentation of large, soluble proteins up to 140 kDa for 1H-detected MAS NMR and 13C DNP NMR - practical aspects

Solution NMR is typically applied to biological systems with molecular weights < 40 kDa whereas magic-angle-spinning (MAS) solid-state NMR traditionally targets very large, oligomeric proteins and complexes exceeding 500 kDa in mass, including fibrils and crystalline protein preparations. Here, we propose that the gap between these size regimes can be filled by the approach presented that enables investigation of large, soluble and fully protonated proteins in the range of 40-140 kDa. As a key...

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0 Replies | 55 Views
NMR solution structure determination of large RNA-protein complexes - ScienceDirect.com
Mar 11, 2024 - 4:18 AM - by nmrlearner
nmrlearner's Avatar NMR solution structure determination of large RNA-protein complexes - ScienceDirect.com

NMR solution structure determination of large RNA-protein complexes ScienceDirect.com Read here
0 Replies | 86 Views
[NMR paper] Genetically encoded site-specific (19)F unnatural amino acid incorporation in V. natriegens for in-cell NMR analysis
Mar 11, 2024 - 4:18 AM - by nmrlearner
nmrlearner's Avatar Genetically encoded site-specific (19)F unnatural amino acid incorporation in V. natriegens for in-cell NMR analysis

Nuclear magnetic resonance (NMR) spectroscopy NMR is a well-established technique for probing protein structure, dynamics and conformational changes. Taking advantage of the high signal sensitivity and broad chemical shift range of ^(19)F nuclei, ^(19)F NMR has been applied to investigate protein function at atomic resolution. In this report, we extend the unnatural amino acid site-specific incorporation into V. natriegens, an alternate protein expression system. The unnatural amino acid...

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0 Replies | 111 Views
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