Articles | Volume 4, issue 2
https://doi.org/10.5194/mr-4-187-2023
© Author(s) 2023. 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-4-187-2023
© Author(s) 2023. This work is distributed under
the Creative Commons Attribution 4.0 License.
the Creative Commons Attribution 4.0 License.
Cell-free synthesis of proteins with selectively 13C-labelled methyl groups from inexpensive precursors
Damian Van Raad
Research School of Chemistry, Australian National University, Canberra, ACT 2601, Australia
Gottfried Otting
CORRESPONDING AUTHOR
Research School of Chemistry, Australian National University, Canberra, ACT 2601, Australia
ARC Centre of Excellence for Innovations in Peptide & Protein
Science, Research School of Chemistry, Australian National University,
Canberra, ACT 2601, Australia
Thomas Huber
CORRESPONDING AUTHOR
Research School of Chemistry, Australian National University, Canberra, ACT 2601, Australia
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Yi Jiun Tan, Elwy H. Abdelkader, Iresha D. Herath, Ansis Maleckis, and Gottfried Otting
Magn. Reson. Discuss., https://doi.org/10.5194/mr-2025-4, https://doi.org/10.5194/mr-2025-4, 2025
Preprint under review for MR
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A protein was produced, where a single amino acid type was substituted globally by a fluorinated analogue, delivering multiple sites for interrogation by 19F-NMR 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.
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
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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.
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
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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.
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.
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
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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.
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: (Bio)Chemistry
Paramagnetic relaxivity of delocalized long-lived states of protons in chains of CH2 groups
Aiky Razanahoera, Anna Sonnefeld, Geoffrey Bodenhausen, and Kirill Sheberstov
Magn. Reson., 4, 47–56, https://doi.org/10.5194/mr-4-47-2023, https://doi.org/10.5194/mr-4-47-2023, 2023
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In this work, we study the relaxivity of long-lived states (LLSs) excited across two to three CH2 groups in four different compounds and find that a commonly used polarising agent in dissolution dynamic nuclear polarization (d-DNP) does not enhance relaxation significantly. This result is important for the future implementation of d-DNP hyperpolarization of LLS.
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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.
A novel cell-free protein synthesis system called eCells produces amino acids based on specific...