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NMR Assignment of Methyl Groups in Immobilized Proteins Using Multiple-Bond (13)C Homonuclear Transfers, Proton Detection, and Very Fast MAS
In nuclear magnetic resonance spectroscopy of proteins, methyl protons play a particular role as extremely sensitive reporters on dynamics, allosteric effects, and protein–protein interactions, accessible even in high-molecular-weight systems approaching 1 MDa. The notorious issue of their chemical...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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Frontiers Media S.A.
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9002630/ https://www.ncbi.nlm.nih.gov/pubmed/35425812 http://dx.doi.org/10.3389/fmolb.2022.828785 |
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author | Paluch, Piotr Augustyniak, Rafal Org, Mai-Liis Vanatalu, Kalju Kaldma, Ats Samoson, Ago Stanek, Jan |
author_facet | Paluch, Piotr Augustyniak, Rafal Org, Mai-Liis Vanatalu, Kalju Kaldma, Ats Samoson, Ago Stanek, Jan |
author_sort | Paluch, Piotr |
collection | PubMed |
description | In nuclear magnetic resonance spectroscopy of proteins, methyl protons play a particular role as extremely sensitive reporters on dynamics, allosteric effects, and protein–protein interactions, accessible even in high-molecular-weight systems approaching 1 MDa. The notorious issue of their chemical shift assignment is addressed here by a joint use of solid-state (1)H-detected methods at very fast (nearly 100 kHz) magic-angle spinning, partial deuteration, and high-magnetic fields. The suitability of a series of RF schemes is evaluated for the efficient coherence transfer across entire (13)C side chains of methyl-containing residues, which is key for establishing connection between methyl and backbone (1)H resonances. The performance of ten methods for recoupling of either isotropic (13)C–(13)C scalar or anisotropic dipolar interactions (five variants of TOBSY, FLOPSY, DIPSI, WALTZ, RFDR, and DREAM) is evaluated experimentally at two state-of-the-art magic-angle spinning (55 and 94.5 kHz) and static magnetic field conditions (18.8 and 23.5 T). Model isotopically labeled compounds (alanine and Met-Leu-Phe tripeptide) and ILV-methyl and amide-selectively protonated, and otherwise deuterated chicken α-spectrin SH3 protein are used as convenient reference systems. Spin dynamics simulations in SIMPSON are performed to determine optimal parameters of these RF schemes, up to recently experimentally attained spinning frequencies (200 kHz) and B (0) field strengths (28.2 T). The concept of linearization of (13)C side chain by appropriate isotope labeling is revisited and showed to significantly increase sensitivity of methyl-to-backbone correlations. A resolution enhancement provided by 4D spectroscopy with non-uniform (sparse) sampling is demonstrated to remove ambiguities in simultaneous resonance assignment of methyl proton and carbon chemical shifts. |
format | Online Article Text |
id | pubmed-9002630 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-90026302022-04-13 NMR Assignment of Methyl Groups in Immobilized Proteins Using Multiple-Bond (13)C Homonuclear Transfers, Proton Detection, and Very Fast MAS Paluch, Piotr Augustyniak, Rafal Org, Mai-Liis Vanatalu, Kalju Kaldma, Ats Samoson, Ago Stanek, Jan Front Mol Biosci Molecular Biosciences In nuclear magnetic resonance spectroscopy of proteins, methyl protons play a particular role as extremely sensitive reporters on dynamics, allosteric effects, and protein–protein interactions, accessible even in high-molecular-weight systems approaching 1 MDa. The notorious issue of their chemical shift assignment is addressed here by a joint use of solid-state (1)H-detected methods at very fast (nearly 100 kHz) magic-angle spinning, partial deuteration, and high-magnetic fields. The suitability of a series of RF schemes is evaluated for the efficient coherence transfer across entire (13)C side chains of methyl-containing residues, which is key for establishing connection between methyl and backbone (1)H resonances. The performance of ten methods for recoupling of either isotropic (13)C–(13)C scalar or anisotropic dipolar interactions (five variants of TOBSY, FLOPSY, DIPSI, WALTZ, RFDR, and DREAM) is evaluated experimentally at two state-of-the-art magic-angle spinning (55 and 94.5 kHz) and static magnetic field conditions (18.8 and 23.5 T). Model isotopically labeled compounds (alanine and Met-Leu-Phe tripeptide) and ILV-methyl and amide-selectively protonated, and otherwise deuterated chicken α-spectrin SH3 protein are used as convenient reference systems. Spin dynamics simulations in SIMPSON are performed to determine optimal parameters of these RF schemes, up to recently experimentally attained spinning frequencies (200 kHz) and B (0) field strengths (28.2 T). The concept of linearization of (13)C side chain by appropriate isotope labeling is revisited and showed to significantly increase sensitivity of methyl-to-backbone correlations. A resolution enhancement provided by 4D spectroscopy with non-uniform (sparse) sampling is demonstrated to remove ambiguities in simultaneous resonance assignment of methyl proton and carbon chemical shifts. Frontiers Media S.A. 2022-03-29 /pmc/articles/PMC9002630/ /pubmed/35425812 http://dx.doi.org/10.3389/fmolb.2022.828785 Text en Copyright © 2022 Paluch, Augustyniak, Org, Vanatalu, Kaldma, Samoson and Stanek. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
spellingShingle | Molecular Biosciences Paluch, Piotr Augustyniak, Rafal Org, Mai-Liis Vanatalu, Kalju Kaldma, Ats Samoson, Ago Stanek, Jan NMR Assignment of Methyl Groups in Immobilized Proteins Using Multiple-Bond (13)C Homonuclear Transfers, Proton Detection, and Very Fast MAS |
title | NMR Assignment of Methyl Groups in Immobilized Proteins Using Multiple-Bond (13)C Homonuclear Transfers, Proton Detection, and Very Fast MAS |
title_full | NMR Assignment of Methyl Groups in Immobilized Proteins Using Multiple-Bond (13)C Homonuclear Transfers, Proton Detection, and Very Fast MAS |
title_fullStr | NMR Assignment of Methyl Groups in Immobilized Proteins Using Multiple-Bond (13)C Homonuclear Transfers, Proton Detection, and Very Fast MAS |
title_full_unstemmed | NMR Assignment of Methyl Groups in Immobilized Proteins Using Multiple-Bond (13)C Homonuclear Transfers, Proton Detection, and Very Fast MAS |
title_short | NMR Assignment of Methyl Groups in Immobilized Proteins Using Multiple-Bond (13)C Homonuclear Transfers, Proton Detection, and Very Fast MAS |
title_sort | nmr assignment of methyl groups in immobilized proteins using multiple-bond (13)c homonuclear transfers, proton detection, and very fast mas |
topic | Molecular Biosciences |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9002630/ https://www.ncbi.nlm.nih.gov/pubmed/35425812 http://dx.doi.org/10.3389/fmolb.2022.828785 |
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