Site-selective generation of lanthanoid binding sites on proteins using 4-fluoro-2,6-dicyanopyridine
<p>The paramagnetism of a lanthanoid tag site-specifically installed on a protein provides a rich source of structural information accessible by nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR) spectroscopy. Here we report a lanthanoid tag for selective reaction with...
| Main Authors: | , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Copernicus Publications
2022-09-01
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| Series: | Magnetic Resonance |
| Online Access: | https://mr.copernicus.org/articles/3/169/2022/mr-3-169-2022.pdf |
| _version_ | 1798001889408712704 |
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| author | S. Mekkattu Tharayil M. C. Mahawaththa A. Feintuch A. Maleckis S. Ullrich R. Morewood M. J. Maxwell T. Huber C. Nitsche D. Goldfarb G. Otting |
| author_facet | S. Mekkattu Tharayil M. C. Mahawaththa A. Feintuch A. Maleckis S. Ullrich R. Morewood M. J. Maxwell T. Huber C. Nitsche D. Goldfarb G. Otting |
| author_sort | S. Mekkattu Tharayil |
| collection | DOAJ |
| description | <p>The paramagnetism of a lanthanoid tag site-specifically installed on a protein provides a rich source of structural information accessible by
nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR) spectroscopy. Here we report a lanthanoid tag for selective reaction
with cysteine or selenocysteine with formation of a (seleno)thioether bond and a short tether between the lanthanoid ion and the protein backbone. The tag
is assembled on the protein in three steps, comprising (i) reaction with 4-fluoro-2,6-dicyanopyridine (FDCP); (ii) reaction of the cyano groups with
<span class="inline-formula"><i>α</i></span>-cysteine, penicillamine or <span class="inline-formula"><i>β</i></span>-cysteine to complete the lanthanoid chelating moiety; and (iii) titration with a lanthanoid ion. FDCP
reacts much faster with selenocysteine than cysteine, opening a route for selective tagging in the presence of solvent-exposed cysteine residues.
Loaded with <span class="inline-formula">Tb<sup>3+</sup></span> and <span class="inline-formula">Tm<sup>3+</sup></span> ions, pseudocontact shifts were observed in protein NMR spectra, confirming that the tag delivers good
immobilisation of the lanthanoid ion relative to the protein, which was also manifested in residual dipolar couplings. Completion of the tag with
different 1,2-aminothiol compounds resulted in different magnetic susceptibility tensors. In addition, the tag proved suitable for measuring
distance distributions in double electron–electron resonance experiments after titration with <span class="inline-formula">Gd<sup>3+</sup></span> ions.</p> |
| first_indexed | 2024-04-11T11:43:20Z |
| format | Article |
| id | doaj.art-6b5aa5e31f6144deb381d559ef943902 |
| institution | Directory Open Access Journal |
| issn | 2699-0016 |
| language | English |
| last_indexed | 2024-04-11T11:43:20Z |
| publishDate | 2022-09-01 |
| publisher | Copernicus Publications |
| record_format | Article |
| series | Magnetic Resonance |
| spelling | doaj.art-6b5aa5e31f6144deb381d559ef9439022022-12-22T04:25:45ZengCopernicus PublicationsMagnetic Resonance2699-00162022-09-01316918210.5194/mr-3-169-2022Site-selective generation of lanthanoid binding sites on proteins using 4-fluoro-2,6-dicyanopyridineS. Mekkattu Tharayil0M. C. Mahawaththa1A. Feintuch2A. Maleckis3S. Ullrich4R. Morewood5M. J. Maxwell6T. Huber7C. Nitsche8D. Goldfarb9G. Otting10Research School of Chemistry, Australian National University, Canberra, ACT 2601, AustraliaARC Centre of Excellence for Innovations in Peptide & Protein Science, Research School of Chemistry, Australian National University, Canberra, ACT 2601, AustraliaDepartment of Chemical Physics, Weizmann Institute of Science, Rehovot 76100, IsraelLatvian Institute of Organic Synthesis, Aizkraukles 21, 1006 Riga, LatviaResearch School of Chemistry, Australian National University, Canberra, ACT 2601, AustraliaResearch School of Chemistry, Australian National University, Canberra, ACT 2601, AustraliaARC Centre of Excellence for Innovations in Peptide & Protein Science, Research School of Chemistry, Australian National University, Canberra, ACT 2601, AustraliaResearch School of Chemistry, Australian National University, Canberra, ACT 2601, AustraliaResearch School of Chemistry, Australian National University, Canberra, ACT 2601, AustraliaDepartment of Chemical Physics, Weizmann Institute of Science, Rehovot 76100, IsraelARC Centre of Excellence for Innovations in Peptide & Protein Science, Research School of Chemistry, Australian National University, Canberra, ACT 2601, Australia<p>The paramagnetism of a lanthanoid tag site-specifically installed on a protein provides a rich source of structural information accessible by nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR) spectroscopy. Here we report a lanthanoid tag for selective reaction with cysteine or selenocysteine with formation of a (seleno)thioether bond and a short tether between the lanthanoid ion and the protein backbone. The tag is assembled on the protein in three steps, comprising (i) reaction with 4-fluoro-2,6-dicyanopyridine (FDCP); (ii) reaction of the cyano groups with <span class="inline-formula"><i>α</i></span>-cysteine, penicillamine or <span class="inline-formula"><i>β</i></span>-cysteine to complete the lanthanoid chelating moiety; and (iii) titration with a lanthanoid ion. FDCP reacts much faster with selenocysteine than cysteine, opening a route for selective tagging in the presence of solvent-exposed cysteine residues. Loaded with <span class="inline-formula">Tb<sup>3+</sup></span> and <span class="inline-formula">Tm<sup>3+</sup></span> ions, pseudocontact shifts were observed in protein NMR spectra, confirming that the tag delivers good immobilisation of the lanthanoid ion relative to the protein, which was also manifested in residual dipolar couplings. Completion of the tag with different 1,2-aminothiol compounds resulted in different magnetic susceptibility tensors. In addition, the tag proved suitable for measuring distance distributions in double electron–electron resonance experiments after titration with <span class="inline-formula">Gd<sup>3+</sup></span> ions.</p>https://mr.copernicus.org/articles/3/169/2022/mr-3-169-2022.pdf |
| spellingShingle | S. Mekkattu Tharayil M. C. Mahawaththa A. Feintuch A. Maleckis S. Ullrich R. Morewood M. J. Maxwell T. Huber C. Nitsche D. Goldfarb G. Otting Site-selective generation of lanthanoid binding sites on proteins using 4-fluoro-2,6-dicyanopyridine Magnetic Resonance |
| title | Site-selective generation of lanthanoid binding sites on proteins using 4-fluoro-2,6-dicyanopyridine |
| title_full | Site-selective generation of lanthanoid binding sites on proteins using 4-fluoro-2,6-dicyanopyridine |
| title_fullStr | Site-selective generation of lanthanoid binding sites on proteins using 4-fluoro-2,6-dicyanopyridine |
| title_full_unstemmed | Site-selective generation of lanthanoid binding sites on proteins using 4-fluoro-2,6-dicyanopyridine |
| title_short | Site-selective generation of lanthanoid binding sites on proteins using 4-fluoro-2,6-dicyanopyridine |
| title_sort | site selective generation of lanthanoid binding sites on proteins using 4 fluoro 2 6 dicyanopyridine |
| url | https://mr.copernicus.org/articles/3/169/2022/mr-3-169-2022.pdf |
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