Site-Specific Introduction of Alanines for the Nuclear Magnetic Resonance Investigation of Low-Complexity Regions and Large Biomolecular Assemblies
Nuclear magnetic resonance (NMR) studies of large biomolecular machines and highly repetitive proteins remain challenging due to the difficulty of assigning frequencies to individual nuclei. Here, we present an efficient strategy to address this challenge by engineering a Pyrococcus horikoshii tRNA/...
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| Published in: | ACS chemical biology Vol. 18; no. 9; pp. 2039 - 2049 |
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| Main Authors: | , , , , , , , , |
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15-09-2023
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| Abstract | Nuclear magnetic resonance (NMR) studies of large biomolecular machines and highly repetitive proteins remain challenging due to the difficulty of assigning frequencies to individual nuclei. Here, we present an efficient strategy to address this challenge by engineering a Pyrococcus horikoshii tRNA/alanyl-tRNA synthetase pair that enables the incorporation of up to three isotopically labeled alanine residues in a site-specific manner using in vitro protein expression. The general applicability of this approach for NMR assignment has been demonstrated by introducing isotopically labeled alanines into four distinct proteins: huntingtin exon-1, HMA8 ATPase, the 300 kDa molecular chaperone ClpP, and the alanine-rich Phox2B transcription factor. For large protein assemblies, our labeling approach enabled unambiguous assignments while avoiding potential artifacts induced by site-specific mutations. When applied to Phox2B, which contains two poly-alanine tracts of nine and twenty alanines, we observed that the helical stability is strongly dependent on the homorepeat length. The capacity to selectively introduce alanines with distinct labeling patterns is a powerful tool to probe structure and dynamics of challenging biomolecular systems. |
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| AbstractList | Nuclear magnetic resonance (NMR) studies of large biomolecular machines and highly repetitive proteins remain challenging due to the difficulty of assigning frequencies to individual nuclei. Here, we present an efficient strategy to address this challenge by engineering a Pyrococcus horikoshii tRNA/alanyl-tRNA synthetase pair that enables the incorporation of up to three isotopically labeled alanine residues in a site-specific manner using in vitro protein expression. The general applicability of this approach for NMR assignment has been demonstrated by introducing isotopically labeled alanines into four distinct proteins: huntingtin exon-1, HMA8 ATPase, the 300 kDa molecular chaperone ClpP, and the alanine-rich Phox2B transcription factor. For large protein assemblies, our labeling approach enabled unambiguous assignments while avoiding potential artifacts induced by site-specific mutations. When applied to Phox2B, which contains two poly-alanine tracts of nine and twenty alanines, we observed that the helical stability is strongly dependent on the homorepeat length. The capacity to selectively introduce alanines with distinct labeling patterns is a powerful tool to probe structure and dynamics of challenging biomolecular systems.Nuclear magnetic resonance (NMR) studies of large biomolecular machines and highly repetitive proteins remain challenging due to the difficulty of assigning frequencies to individual nuclei. Here, we present an efficient strategy to address this challenge by engineering a Pyrococcus horikoshii tRNA/alanyl-tRNA synthetase pair that enables the incorporation of up to three isotopically labeled alanine residues in a site-specific manner using in vitro protein expression. The general applicability of this approach for NMR assignment has been demonstrated by introducing isotopically labeled alanines into four distinct proteins: huntingtin exon-1, HMA8 ATPase, the 300 kDa molecular chaperone ClpP, and the alanine-rich Phox2B transcription factor. For large protein assemblies, our labeling approach enabled unambiguous assignments while avoiding potential artifacts induced by site-specific mutations. When applied to Phox2B, which contains two poly-alanine tracts of nine and twenty alanines, we observed that the helical stability is strongly dependent on the homorepeat length. The capacity to selectively introduce alanines with distinct labeling patterns is a powerful tool to probe structure and dynamics of challenging biomolecular systems. Nuclear magnetic resonance (NMR) studies of large biomolecular machines and highly repetitive proteins remain challenging due to the difficulty of assigning frequencies to individual nuclei. Here, we present an efficient strategy to address this challenge by engineering a Pyrococcus horikoshii tRNA/alanyl-tRNA synthetase pair that enables the incorporation of up to three isotopically labeled alanine residues in a site-specific manner using in vitro protein expression. The general applicability of this approach for NMR assignment has been demonstrated by introducing isotopically labeled alanines into four distinct proteins: huntingtin exon-1, HMA8 ATPase, the 300 kDa molecular chaperone ClpP, and the alanine-rich Phox2B transcription factor. For large protein assemblies, our labeling approach enabled unambiguous assignments while avoiding potential artifacts induced by site-specific mutations. When applied to Phox2B, which contains two poly-alanine tracts of nine and twenty alanines, we observed that the helical stability is strongly dependent on the homorepeat length. The capacity to selectively introduce alanines with distinct labeling patterns is a powerful tool to probe structure and dynamics of challenging biomolecular systems. |
| Author | Elena-Real, Carlos A. Imbert, Lionel Fournet, Aurélie Sibille, Nathalie Morató, Anna Allemand, Frédéric Bernadó, Pau Boisbouvier, Jérôme Urbanek, Annika |
| AuthorAffiliation | Univ. Grenoble Alpes, CNRS, CEA, Institut de Biologie Structurale (IBS) |
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| Author_xml | – sequence: 1 givenname: Carlos A. surname: Elena-Real fullname: Elena-Real, Carlos A. – sequence: 2 givenname: Annika surname: Urbanek fullname: Urbanek, Annika – sequence: 3 givenname: Lionel surname: Imbert fullname: Imbert, Lionel organization: Univ. Grenoble Alpes, CNRS, CEA, Institut de Biologie Structurale (IBS) – sequence: 4 givenname: Anna surname: Morató fullname: Morató, Anna – sequence: 5 givenname: Aurélie surname: Fournet fullname: Fournet, Aurélie – sequence: 6 givenname: Frédéric surname: Allemand fullname: Allemand, Frédéric – sequence: 7 givenname: Nathalie surname: Sibille fullname: Sibille, Nathalie – sequence: 8 givenname: Jérôme orcidid: 0000-0003-3278-3639 surname: Boisbouvier fullname: Boisbouvier, Jérôme email: jerome.boisbouvier@ibs.fr organization: Univ. Grenoble Alpes, CNRS, CEA, Institut de Biologie Structurale (IBS) – sequence: 9 givenname: Pau orcidid: 0000-0001-7395-5922 surname: Bernadó fullname: Bernadó, Pau email: pau.bernado@cbs.cnrs.fr |
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| Title | Site-Specific Introduction of Alanines for the Nuclear Magnetic Resonance Investigation of Low-Complexity Regions and Large Biomolecular Assemblies |
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