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Phosphoserine for the generation of lanthanide-binding sites on proteins for paramagnetic nuclear magnetic resonance spectroscopy
Pseudocontact shifts (PCSs) generated by paramagnetic lanthanide ions provide valuable long-range structural information in nuclear magnetic resonance (NMR) spectroscopic analyses of biological macromolecules such as proteins, but labelling proteins site-specifically with a single lanthanide ion rem...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Copernicus GmbH
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10539748/ https://www.ncbi.nlm.nih.gov/pubmed/37904776 http://dx.doi.org/10.5194/mr-2-1-2021 |
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author | Mekkattu Tharayil, Sreelakshmi Mahawaththa, Mithun Chamikara Loh, Choy-Theng Adekoya, Ibidolapo Otting, Gottfried |
author_facet | Mekkattu Tharayil, Sreelakshmi Mahawaththa, Mithun Chamikara Loh, Choy-Theng Adekoya, Ibidolapo Otting, Gottfried |
author_sort | Mekkattu Tharayil, Sreelakshmi |
collection | PubMed |
description | Pseudocontact shifts (PCSs) generated by paramagnetic lanthanide ions provide valuable long-range structural information in nuclear magnetic resonance (NMR) spectroscopic analyses of biological macromolecules such as proteins, but labelling proteins site-specifically with a single lanthanide ion remains an ongoing challenge, especially for proteins that are not suitable for ligation with cysteine-reactive lanthanide complexes. We show that a specific lanthanide-binding site can be installed on proteins by incorporation of phosphoserine in conjunction with other negatively charged residues, such as aspartate, glutamate or a second phosphoserine residue. The close proximity of the binding sites to the protein backbone leads to good immobilization of the lanthanide ion, as evidenced by the excellent quality of fits between experimental PCSs and PCSs calculated with a single magnetic susceptibility anisotropy ( [Formula: see text] tensor. An improved two-plasmid system was designed to enhance the yields of proteins with genetically encoded phosphoserine, and good lanthanide ion affinities were obtained when the side chains of the phosphoserine and aspartate residues are not engaged in salt bridges, although the presence of too many negatively charged residues in close proximity can also lead to unfolding of the protein. In view of the quality of the [Formula: see text] tensors that can be obtained from lanthanide-binding sites generated by site-specific incorporation of phosphoserine, this method presents an attractive tool for generating PCSs in stable proteins, particularly as it is independent of cysteine residues. |
format | Online Article Text |
id | pubmed-10539748 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | Copernicus GmbH |
record_format | MEDLINE/PubMed |
spelling | pubmed-105397482023-10-30 Phosphoserine for the generation of lanthanide-binding sites on proteins for paramagnetic nuclear magnetic resonance spectroscopy Mekkattu Tharayil, Sreelakshmi Mahawaththa, Mithun Chamikara Loh, Choy-Theng Adekoya, Ibidolapo Otting, Gottfried Magn Reson (Gott) Research Article Pseudocontact shifts (PCSs) generated by paramagnetic lanthanide ions provide valuable long-range structural information in nuclear magnetic resonance (NMR) spectroscopic analyses of biological macromolecules such as proteins, but labelling proteins site-specifically with a single lanthanide ion remains an ongoing challenge, especially for proteins that are not suitable for ligation with cysteine-reactive lanthanide complexes. We show that a specific lanthanide-binding site can be installed on proteins by incorporation of phosphoserine in conjunction with other negatively charged residues, such as aspartate, glutamate or a second phosphoserine residue. The close proximity of the binding sites to the protein backbone leads to good immobilization of the lanthanide ion, as evidenced by the excellent quality of fits between experimental PCSs and PCSs calculated with a single magnetic susceptibility anisotropy ( [Formula: see text] tensor. An improved two-plasmid system was designed to enhance the yields of proteins with genetically encoded phosphoserine, and good lanthanide ion affinities were obtained when the side chains of the phosphoserine and aspartate residues are not engaged in salt bridges, although the presence of too many negatively charged residues in close proximity can also lead to unfolding of the protein. In view of the quality of the [Formula: see text] tensors that can be obtained from lanthanide-binding sites generated by site-specific incorporation of phosphoserine, this method presents an attractive tool for generating PCSs in stable proteins, particularly as it is independent of cysteine residues. Copernicus GmbH 2021-01-06 /pmc/articles/PMC10539748/ /pubmed/37904776 http://dx.doi.org/10.5194/mr-2-1-2021 Text en Copyright: © 2021 Sreelakshmi Mekkattu Tharayil et al. https://creativecommons.org/licenses/by/4.0/This work is licensed under the Creative Commons Attribution 4.0 International License. To view a copy of this licence, visit https://creativecommons.org/licenses/by/4.0/ |
spellingShingle | Research Article Mekkattu Tharayil, Sreelakshmi Mahawaththa, Mithun Chamikara Loh, Choy-Theng Adekoya, Ibidolapo Otting, Gottfried Phosphoserine for the generation of lanthanide-binding sites on proteins for paramagnetic nuclear magnetic resonance spectroscopy |
title | Phosphoserine for the generation of lanthanide-binding sites on proteins for paramagnetic nuclear magnetic resonance spectroscopy |
title_full | Phosphoserine for the generation of lanthanide-binding sites on proteins for paramagnetic nuclear magnetic resonance spectroscopy |
title_fullStr | Phosphoserine for the generation of lanthanide-binding sites on proteins for paramagnetic nuclear magnetic resonance spectroscopy |
title_full_unstemmed | Phosphoserine for the generation of lanthanide-binding sites on proteins for paramagnetic nuclear magnetic resonance spectroscopy |
title_short | Phosphoserine for the generation of lanthanide-binding sites on proteins for paramagnetic nuclear magnetic resonance spectroscopy |
title_sort | phosphoserine for the generation of lanthanide-binding sites on proteins for paramagnetic nuclear magnetic resonance spectroscopy |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10539748/ https://www.ncbi.nlm.nih.gov/pubmed/37904776 http://dx.doi.org/10.5194/mr-2-1-2021 |
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