Articles | Volume 2, issue 1
https://doi.org/10.5194/mr-2-1-2021
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the Creative Commons Attribution 4.0 License.
Special issue:
https://doi.org/10.5194/mr-2-1-2021
© Author(s) 2021. This work is distributed under
the Creative Commons Attribution 4.0 License.
the Creative Commons Attribution 4.0 License.
Research article
| 
06 Jan 2021
Research article |
| 06 Jan 2021
| 06 Jan 2021
Phosphoserine for the generation of lanthanide-binding sites on proteins for paramagnetic nuclear magnetic resonance spectroscopy
Sreelakshmi Mekkattu Tharayil
ARC Centre of Excellence for Innovations in Peptide and Protein
Science, Research School of Chemistry, Australian National University,
Canberra ACT 2601, Australia
Mithun Chamikara Mahawaththa
ARC Centre of Excellence for Innovations in Peptide and Protein
Science, Research School of Chemistry, Australian National University,
Canberra ACT 2601, Australia
Choy-Theng Loh
ARC Centre of Excellence for Innovations in Peptide and Protein
Science, Research School of Chemistry, Australian National University,
Canberra ACT 2601, Australia
present address: Hangzhou Wayland Bioscience Co. Ltd, Hangzhou
310030, PR China
Ibidolapo Adekoya
ARC Centre of Excellence for Innovations in Peptide and Protein
Science, Research School of Chemistry, Australian National University,
Canberra ACT 2601, Australia
Gottfried Otting
CORRESPONDING AUTHOR
ARC Centre of Excellence for Innovations in Peptide and Protein
Science, Research School of Chemistry, Australian National University,
Canberra ACT 2601, Australia
Related authors
Sreelakshmi Mekkattu Tharayil, Mithun C. Mahawaththa, Akiva Feintuch, Ansis Maleckis, Sven Ullrich, Richard Morewood, Michael J. Maxwell, Thomas Huber, Christoph Nitsche, Daniella Goldfarb, and Gottfried Otting
Magn. Reson., 3, 169–182, https://doi.org/10.5194/mr-3-169-2022, https://doi.org/10.5194/mr-3-169-2022, 2022
Short summary
Short summary
Having shown that tagging a protein at a single site with different lanthanoid complexes delivers outstanding structural information at a selected site of a protein (such as active sites and ligand binding sites), we now present a simple way by which different lanthanoid complexes can be assembled on a highly solvent-exposed cysteine residue. Furthermore, the chemical assembly is selective for selenocysteine, if a selenocysteine residue can be introduced into the protein of interest.
Yi Jiun Tan, Elwy H. Abdelkader, Iresha D. Herath, Ansis Maleckis, and Gottfried Otting
Magn. Reson., 6, 131–142, https://doi.org/10.5194/mr-6-131-2025, https://doi.org/10.5194/mr-6-131-2025, 2025
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A protein is produced where a single amino acid type is substituted globally by a fluorinated analogue. Through-space fluorine–fluorine contacts are observed by 19F NMR (nuclear magnetic resonance) spectroscopy. Substitution of methyl groups by CH2F groups yields outstanding spectral resolution with minimal structural perturbation of the protein. Our work identifies the γ-gauche effect as the main reason for the spectral dispersion.
Damian Van Raad, Gottfried Otting, and Thomas Huber
Magn. Reson., 4, 187–197, https://doi.org/10.5194/mr-4-187-2023, https://doi.org/10.5194/mr-4-187-2023, 2023
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Short summary
A novel cell-free protein synthesis system called eCells produces amino acids based on specific isotopes using low-cost precursors. The system selectively labels methyl groups, i.e valine and leucine, with high efficiency. eCells achieve high levels of 13C incorporation and deuteration in protein preparations, making them suitable for NMR experiments of large protein complexes. They are easy to prepare, can be scaled up in volume and are a promising tool for protein production and NMR studies.
Sreelakshmi Mekkattu Tharayil, Mithun C. Mahawaththa, Akiva Feintuch, Ansis Maleckis, Sven Ullrich, Richard Morewood, Michael J. Maxwell, Thomas Huber, Christoph Nitsche, Daniella Goldfarb, and Gottfried Otting
Magn. Reson., 3, 169–182, https://doi.org/10.5194/mr-3-169-2022, https://doi.org/10.5194/mr-3-169-2022, 2022
Short summary
Short summary
Having shown that tagging a protein at a single site with different lanthanoid complexes delivers outstanding structural information at a selected site of a protein (such as active sites and ligand binding sites), we now present a simple way by which different lanthanoid complexes can be assembled on a highly solvent-exposed cysteine residue. Furthermore, the chemical assembly is selective for selenocysteine, if a selenocysteine residue can be introduced into the protein of interest.
Henry W. Orton, Elwy H. Abdelkader, Lydia Topping, Stephen J. Butler, and Gottfried Otting
Magn. Reson., 3, 65–76, https://doi.org/10.5194/mr-3-65-2022, https://doi.org/10.5194/mr-3-65-2022, 2022
Short summary
Short summary
Installing a tag containing a paramagnetic metal ion on a protein can lead to large changes (pseudocontact shifts) in the resonances observed in NMR spectra. These are easily measured and contain valuable long-range structural information. The present work shows that a single tagging site furnished with different tags can be sufficient to localise atoms in proteins with high accuracy. In fact, this strategy works almost as well as the same number of tags distributed over multiple tagging sites.
Henry W. Orton, Iresha D. Herath, Ansis Maleckis, Shereen Jabar, Monika Szabo, Bim Graham, Colum Breen, Lydia Topping, Stephen J. Butler, and Gottfried Otting
Magn. Reson., 3, 1–13, https://doi.org/10.5194/mr-3-1-2022, https://doi.org/10.5194/mr-3-1-2022, 2022
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This paper explores a method for determining the solution structure of a solvent-exposed polypeptide segment (the L3 loop), which is next to the active site of the penicillin-degrading enzyme IMP-1. Tagging three different sites on the protein with paramagnetic metal ions allowed positioning of the L3 loop with atomic resolution. It was found that the method was more robust when omitting data obtained with different metal ions if obtained with the same tag at the same tagging site.
Henry William Orton, Thomas Huber, and Gottfried Otting
Magn. Reson., 1, 1–12, https://doi.org/10.5194/mr-1-1-2020, https://doi.org/10.5194/mr-1-1-2020, 2020
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Nuclear magnetic resonance is a technique that allows the measurement of the nanoscale distances between the atoms of a molecule. It is often relied upon for finding the structure of a molecule and how atoms are bonded, but measurements become inaccurate for large distances and therefore for large biological molecules. This research presents a new software for calculating the distances between nuclei and unpaired electrons which offers more accurate long-range distances in biological molecules.
Related subject area
Field: Liquid-state NMR | Topic: Applications – biological macromolecules
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Site-selective generation of lanthanoid binding sites on proteins using 4-fluoro-2,6-dicyanopyridine
Imatinib disassembles the regulatory core of Abelson kinase by binding to its ATP site and not by binding to its myristoyl pocket
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Anomalous amide proton chemical shifts as signatures of hydrogen bonding to aromatic sidechains
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High-affinity tamoxifen analogues retain extensive positional disorder when bound to calmodulin
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Small-molecule inhibitors of the PDZ domain of Dishevelled proteins interrupt Wnt signalling
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Tayeb Kakeshpour, Martin D. Gelenter, Jinfa Ying, and Ad Bax
Magn. Reson. Discuss., https://doi.org/10.5194/mr-2025-5, https://doi.org/10.5194/mr-2025-5, 2025
Revised manuscript accepted for MR
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3D printed sample cells, inserted into standard 5-mm NMR tubes, are demonstrated to offer improved sensitivity for biomolecular NMR when the quantity of material is limiting. By using an ellipsoidal shape, magnetic field homogeneity inside the microcell to first order is insensitive to differences in the magnetic susceptibility of printer resin and solvent. The sample cells are particularly useful at high ionic strength and at ultra-high magnetic fields.
Louis Brigandat, Maëlys Laux, Caroline Marteau, Laura Cole, Anja Böckmann, Lauriane Lecoq, Marie-Laure Fogeron, and Morgane Callon
Magn. Reson., 5, 95–101, https://doi.org/10.5194/mr-5-95-2024, https://doi.org/10.5194/mr-5-95-2024, 2024
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We used NMR to sequentially assign the side-chain resonances of the cytosolic domain of glycoprotein n of the Crimean–Congo hemorrhagic fever virus. The combination of cell-free protein synthesis with high-field NMR and artificial intelligence approaches facilitated a time- and effort-efficient approach. Our results will be harnessed to study the membrane-bound form of the domain and its interactions with virulence factors, which will ultimately help to understand their role in disease.
Sarah Kuschert, Martin Stroet, Yanni Ka-Yan Chin, Anne Claire Conibear, Xinying Jia, Thomas Lee, Christian Reinhard Otto Bartling, Kristian Strømgaard, Peter Güntert, Karl Johan Rosengren, Alan Edward Mark, and Mehdi Mobli
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The 20 genetically encoded amino acids provide the basis for most proteins and peptides that make up the machinery of life. This limited repertoire is vastly expanded by the introduction of non-canonical amino acids (ncAAs). Studying the structure of protein-containing ncAAs requires new computational representations that are compatible with existing modelling software. We have developed an online tool for this to aid future structural studies of this class of complex biopolymer.
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Magn. Reson., 3, 169–182, https://doi.org/10.5194/mr-3-169-2022, https://doi.org/10.5194/mr-3-169-2022, 2022
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Having shown that tagging a protein at a single site with different lanthanoid complexes delivers outstanding structural information at a selected site of a protein (such as active sites and ligand binding sites), we now present a simple way by which different lanthanoid complexes can be assembled on a highly solvent-exposed cysteine residue. Furthermore, the chemical assembly is selective for selenocysteine, if a selenocysteine residue can be introduced into the protein of interest.
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Magn. Reson., 3, 65–76, https://doi.org/10.5194/mr-3-65-2022, https://doi.org/10.5194/mr-3-65-2022, 2022
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Short summary
Installing a tag containing a paramagnetic metal ion on a protein can lead to large changes (pseudocontact shifts) in the resonances observed in NMR spectra. These are easily measured and contain valuable long-range structural information. The present work shows that a single tagging site furnished with different tags can be sufficient to localise atoms in proteins with high accuracy. In fact, this strategy works almost as well as the same number of tags distributed over multiple tagging sites.
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Fluorine NMR was used to study the interaction between a proline-rich peptide and a SH3 domain using 4S- and 4R-fluorinated prolines whose potential as NMR probes has not been exploited yet. We present a comprehensive study addressing several aspects to be considered when using these residues as NMR probes, including relaxation and dynamics. We show that their conformational bias may be used to modulate the kinetics of protein binding to proline-rich motifs.
Christopher A. Waudby and John Christodoulou
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Kumaran Baskaran, Colin W. Wilburn, Jonathan R. Wedell, Leonardus M. I. Koharudin, Eldon L. Ulrich, Adam D. Schuyler, Hamid R. Eghbalnia, Angela M. Gronenborn, and Jeffrey C. Hoch
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Bei Liu, Atul Rangadurai, Honglue Shi, and Hashim M. Al-Hashimi
Magn. Reson., 2, 715–731, https://doi.org/10.5194/mr-2-715-2021, https://doi.org/10.5194/mr-2-715-2021, 2021
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There is growing interest in mapping exchange dynamics between Watson–Crick and Hoogsteen conformations across different DNA contexts. However, current methods are ill-suited for measurements at a large scale because they require isotopically enriched samples. We report that Hoogsteen dynamics can be measured on unlabeled samples using 1H CEST experiments, which have higher throughput and lower cost relative to conventional methods and also provide new insights into Hoogsteen dynamics.
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The overall aim of the study is to provide a basis from which to improve the ability of tamoxifen family drugs to reduce the activity of a secondary target protein, calmodulin, during tumour development. The main conclusion is that the binding of a tamoxifen analogue is quite unlike that of other anti-calmodulin compounds in that two drug molecules bring the two domains of calmodulin into close proximity, but they are not fixed in orientation relative to the protein.
Chih-Ting Huang, Yei-Chen Lai, Szu-Yun Chen, Meng-Ru Ho, Yun-Wei Chiang, and Shang-Te Danny Hsu
Magn. Reson., 2, 375–386, https://doi.org/10.5194/mr-2-375-2021, https://doi.org/10.5194/mr-2-375-2021, 2021
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Trigger factor (TF) is a conserved bacterial molecular chaperone that exists in a monomer–dimer equilibrium in solution. It binds to the ribosome as a monomer to facilitate folding of nascent polypeptide chains. We showed that dimeric TF exhibits distinct domain dynamics and conformational polymorphism and that TF contains multiple substrate binding sites that are only accessible in its monomeric form. The equilibrium of TF in different oligomeric states may serve as a regulatory mechanism.
Nestor Kamdem, Yvette Roske, Dmytro Kovalskyy, Maxim O. Platonov, Oleksii Balinskyi, Annika Kreuchwig, Jörn Saupe, Liang Fang, Anne Diehl, Peter Schmieder, Gerd Krause, Jörg Rademann, Udo Heinemann, Walter Birchmeier, and Hartmut Oschkinat
Magn. Reson., 2, 355–374, https://doi.org/10.5194/mr-2-355-2021, https://doi.org/10.5194/mr-2-355-2021, 2021
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The Wnt signalling pathway plays a major role in prevention of cancer, whereby the protein Dishevelled connects from the transmembrane receptor Frizzled to downstream effectors via its PDZ domain. Here, cycles of chemical synthesis and structural biology are applied to develop PDZ ligands that block the Frizzled–Dishevelled interaction using NMR for screening, in ligand development, and for deriving structure–activity relationships. Cellular reporter assays demonstrate their efficacy.
György Pintér, Katharina F. Hohmann, J. Tassilo Grün, Julia Wirmer-Bartoschek, Clemens Glaubitz, Boris Fürtig, and Harald Schwalbe
Magn. Reson., 2, 291–320, https://doi.org/10.5194/mr-2-291-2021, https://doi.org/10.5194/mr-2-291-2021, 2021
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The folding, refolding and misfolding of biomacromolecules including proteins, DNA and RNA is an important area of biophysical research to understand functional and disease states of a cell. NMR spectroscopy provides detailed insight, with both high time and atomic resolution. These experiments put stringent requirements on signal-to-noise for often irreversible folding reactions. The review describes methodological approaches and highlights key applications.
Francesca Camponeschi, Angelo Gallo, Mario Piccioli, and Lucia Banci
Magn. Reson., 2, 203–221, https://doi.org/10.5194/mr-2-203-2021, https://doi.org/10.5194/mr-2-203-2021, 2021
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The iron–sulfur cluster binding properties of human mitoNEET have been investigated by 1D and 2D 1H paramagnetic NMR spectroscopy. The NMR spectra of both oxidized and reduced mitoNEET are significantly different from those reported previously for other [Fe2S2] proteins. Our findings revealed the unique electronic properties of mitoNEET and suggests that the specific electronic structure of the cluster possibly drives the functional properties of different [Fe2S2] proteins.
Mitsuhiro Takeda, Yohei Miyanoiri, Tsutomu Terauchi, and Masatsune Kainosho
Magn. Reson., 2, 223–237, https://doi.org/10.5194/mr-2-223-2021, https://doi.org/10.5194/mr-2-223-2021, 2021
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Although both the hydrophobic aliphatic chain and hydrophilic ζ-amino group of the lysine side chain presumably contribute to the structures and functions of proteins, the dual nature of the lysine residue has not been fully understood yet, due to the lack of appropriate methods to acquire comprehensive information on its long consecutive methylene chain at the atomic scale. We describe herein a novel strategy to address the current situation using nuclear magnetic resonance spectroscopy.
Xingjian Xu, Igor Dikiy, Matthew R. Evans, Leandro P. Marcelino, and Kevin H. Gardner
Magn. Reson., 2, 63–76, https://doi.org/10.5194/mr-2-63-2021, https://doi.org/10.5194/mr-2-63-2021, 2021
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While most proteins adopt one conformation, several interconvert between two or more very different structures. Knowing how sequence changes and small-molecule binding can control this behavior is essential for both understanding biology and inspiring new “molecular switches” which can control cellular pathways. This work contributes by examining these topics in the ARNT protein, showing that features of both the folded and unfolded states contribute to the interconversion process.
Rubin Dasgupta, Karthick B. S. S. Gupta, Huub J. M. de Groot, and Marcellus Ubbink
Magn. Reson., 2, 15–23, https://doi.org/10.5194/mr-2-15-2021, https://doi.org/10.5194/mr-2-15-2021, 2021
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A method is demonstrated that can help in sequence-specific NMR signal assignment to nuclear spins near a strongly paramagnetic metal in an enzyme. A combination of paramagnetically tailored NMR experiments and second-shell mutagenesis was used to attribute previously observed chemical exchange processes in the active site of laccase to specific histidine ligands. The signals of nuclei close to the metal can be used as spies to unravel the role of motions in the catalytic process.
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Short summary
A new way is presented for creating lanthanide binding sites on proteins using site-specifically introduced phosphoserine residues. The paramagnetic effects of lanthanides generate long-range effects, which contain structural information and are readily measured by NMR spectroscopy. Excellent correlations between experimentally observed and back-calculated pseudocontact shifts attest to very good immobilization of the lanthanide ions relative to the proteins.
A new way is presented for creating lanthanide binding sites on proteins using site-specifically...
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