Articles | Volume 3, issue 2
https://doi.org/10.5194/mr-3-169-2022
© Author(s) 2022. This work is distributed under
the Creative Commons Attribution 4.0 License.
the Creative Commons Attribution 4.0 License.
https://doi.org/10.5194/mr-3-169-2022
© Author(s) 2022. This work is distributed under
the Creative Commons Attribution 4.0 License.
the Creative Commons Attribution 4.0 License.
Research article
| 
13 Sep 2022
Research article |
| 13 Sep 2022
| 13 Sep 2022
Site-selective generation of lanthanoid binding sites on proteins using 4-fluoro-2,6-dicyanopyridine
Sreelakshmi Mekkattu Tharayil
Research School of Chemistry, Australian National University, Canberra, ACT 2601, Australia
Mithun C. Mahawaththa
ARC Centre of Excellence for Innovations in Peptide & Protein Science, Research School of Chemistry, Australian National University, Canberra, ACT 2601, Australia
Akiva Feintuch
Department of Chemical Physics, Weizmann Institute of Science, Rehovot 76100, Israel
Ansis Maleckis
Latvian Institute of Organic Synthesis, Aizkraukles 21, 1006 Riga, Latvia
Sven Ullrich
Research School of Chemistry, Australian National University, Canberra, ACT 2601, Australia
Richard Morewood
Research School of Chemistry, Australian National University, Canberra, ACT 2601, Australia
Michael J. Maxwell
ARC Centre of Excellence for Innovations in Peptide & Protein Science, Research School of Chemistry, Australian National University, Canberra, ACT 2601, Australia
Thomas Huber
Research School of Chemistry, Australian National University, Canberra, ACT 2601, Australia
Christoph Nitsche
Research School of Chemistry, Australian National University, Canberra, ACT 2601, Australia
Daniella Goldfarb
Department of Chemical Physics, Weizmann Institute of Science, Rehovot 76100, Israel
Gottfried Otting
CORRESPONDING AUTHOR
ARC Centre of Excellence for Innovations in Peptide & Protein Science, Research School of Chemistry, Australian National University, Canberra, ACT 2601, Australia
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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.
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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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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.
Thorsten Bahrenberg, Samuel M. Jahn, Akiva Feintuch, Stefan Stoll, and Daniella Goldfarb
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Double electron–electron resonance (DEER) provides information on the structure of proteins by attaching two spin labels to the protein at a well-defined location and measuring the distance between them. The sensitivity of the method in terms of the amount of the protein that is needed for the experiment depends strongly on the relaxation properties of the spin label and the composition of the solvent. We show how to set up the experiment for best sensitivity when the solvent is water (H2O).
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Magn. Reson., 2, 1–13, https://doi.org/10.5194/mr-2-1-2021, https://doi.org/10.5194/mr-2-1-2021, 2021
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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.
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Through a series of DEER measurements on two Gd rulers, with Gd–Gd distances of 2.1 and 6.0 nm, we show that artefacts commonly observed when measuring short distances can be eliminated by avoiding excitation of the central transition by both the pump and observer pulses. By using a wideband induction mode sample holder at 94 GHz, we demonstrate that high-quality DEER measurements will become possible using Gd spin labels at sub-µM concentrations, with implications for in-cell DEER measurements.
Marie Ramirez Cohen, Akiva Feintuch, Daniella Goldfarb, and Shimon Vega
Magn. Reson., 1, 45–57, https://doi.org/10.5194/mr-1-45-2020, https://doi.org/10.5194/mr-1-45-2020, 2020
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DNP is a method used to enhance NMR signals by transferring polarization from radicals to nuclear spins using microwave irradiation. The EPR spectrum of the radical during the irradiation is determined by a process called spectral diffusion and affects the DNP efficiency. This process can be measured by electron–electron double resonance. We explored experimentally and theoretically the contribution of the hyperfine coupling of the 14N nuclei in the nitroxide radical to the spectral diffusion.
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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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Imatinib disassembles the regulatory core of Abelson kinase by binding to its ATP site and not by binding to its myristoyl pocket
Localising nuclear spins by pseudocontact shifts from a single tagging site
Localising individual atoms of tryptophan side chains in the metallo-β-lactamase IMP-1 by pseudocontact shifts from paramagnetic lanthanoid tags at multiple sites
Fluorine NMR study of proline-rich sequences using fluoroprolines
Analysis of conformational exchange processes using methyl-TROSY-based Hahn echo measurements of quadruple-quantum relaxation
Anomalous amide proton chemical shifts as signatures of hydrogen bonding to aromatic sidechains
Rapid assessment of Watson–Crick to Hoogsteen exchange in unlabeled DNA duplexes using high-power SELOPE imino 1H CEST
High-affinity tamoxifen analogues retain extensive positional disorder when bound to calmodulin
Structural polymorphism and substrate promiscuity of a ribosome-associated molecular chaperone
Small-molecule inhibitors of the PDZ domain of Dishevelled proteins interrupt Wnt signalling
Real-time nuclear magnetic resonance spectroscopy in the study of biomolecular kinetics and dynamics
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Conformational features and ionization states of Lys side chains in a protein studied using the stereo-array isotope labeling (SAIL) method
Fragile protein folds: sequence and environmental factors affecting the equilibrium of two interconverting, stably folded protein conformations
Towards resolving the complex paramagnetic nuclear magnetic resonance (NMR) spectrum of small laccase: assignments of resonances to residue-specific nuclei
Phosphoserine for the generation of lanthanide-binding sites on proteins for paramagnetic nuclear magnetic resonance spectroscopy
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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We show here that binding of the anticancer drug imatinib to the ATP site of Abelson kinase and not binding to its allosteric site coincides with the opening of the kinase regulatory core at nanomolar concentrations. This has implications for the understanding of Abelson’s kinase regulation and activity during medication as well as for the design of new Abelson kinase inhibitors.
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Davy Sinnaeve, Abir Ben Bouzayene, Emile Ottoy, Gert-Jan Hofman, Eva Erdmann, Bruno Linclau, Ilya Kuprov, José C. Martins, Vladimir Torbeev, and Bruno Kieffer
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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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Chih-Ting Huang, Yei-Chen Lai, Szu-Yun Chen, Meng-Ru Ho, Yun-Wei Chiang, and Shang-Te Danny Hsu
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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
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György Pintér, Katharina F. Hohmann, J. Tassilo Grün, Julia Wirmer-Bartoschek, Clemens Glaubitz, Boris Fürtig, and Harald Schwalbe
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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.
Sreelakshmi Mekkattu Tharayil, Mithun Chamikara Mahawaththa, Choy-Theng Loh, Ibidolapo Adekoya, and Gottfried Otting
Magn. Reson., 2, 1–13, https://doi.org/10.5194/mr-2-1-2021, https://doi.org/10.5194/mr-2-1-2021, 2021
Short summary
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.
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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.
Having shown that tagging a protein at a single site with different lanthanoid complexes...
