Pulse EPR-enabled interpretation of scarce pseudocontact shifts induced by lanthanide binding tags

Elwy H Abdelkader, Xuejun Yao, Akiva Feintuch, Luke A Adams, Luigi Aurelio, Bimbil Graham, Daniella Goldfarb, Gottfried Otting

Research output: Contribution to journalArticleResearchpeer-review

Abstract

Pseudocontact shifts (PCS) induced by tags loaded with paramagnetic lanthanide ions provide powerful long-range structure information, provided the location of the metal ion relative to the target protein is known. Usually, the metal position is determined by fitting the magnetic susceptibility anisotropy (Δχ) tensor to the 3D structure of the protein in an 8-parameter fit, which requires a large set of PCSs to be reliable. In an alternative approach, we used multiple Gd3+-Gd3+ distances measured by double electron-electron resonance (DEER) experiments to define the metal position, allowing Δχ-tensor determinations from more robust 5-parameter fits that can be performed with a relatively sparse set of PCSs. Using this approach with the 32 kDa E. coli aspartate/glutamate binding protein (DEBP), we demonstrate a structural transition between substrate-bound and substrate-free DEBP, supported by PCSs generated by C3-Tm3+ and C3-Tb3+ tags attached to a genetically encoded p-azidophenylalanine residue. The significance of small PCSs was magnified by considering the difference between the chemical shifts measured with Tb3+ and Tm3+ rather than involving a diamagnetic reference. The integrative sparse data approach developed in this work makes poorly soluble proteins of limited stability amenable to structural studies in solution, without having to rely on cysteine mutations for tag attachment.

Original languageEnglish
Pages (from-to)39-51
Number of pages13
JournalJournal of Biomolecular NMR
Volume64
Issue number1
DOIs
Publication statusPublished - 2016

Keywords

  • Double electron-electron resonance
  • E. coli aspartate/glutamate binding protein
  • Integrative structural biology
  • Lanthanide tag
  • Pseudocontact shift

Cite this

Abdelkader, Elwy H ; Yao, Xuejun ; Feintuch, Akiva ; Adams, Luke A ; Aurelio, Luigi ; Graham, Bimbil ; Goldfarb, Daniella ; Otting, Gottfried. / Pulse EPR-enabled interpretation of scarce pseudocontact shifts induced by lanthanide binding tags. In: Journal of Biomolecular NMR. 2016 ; Vol. 64, No. 1. pp. 39-51.
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abstract = "Pseudocontact shifts (PCS) induced by tags loaded with paramagnetic lanthanide ions provide powerful long-range structure information, provided the location of the metal ion relative to the target protein is known. Usually, the metal position is determined by fitting the magnetic susceptibility anisotropy (Δχ) tensor to the 3D structure of the protein in an 8-parameter fit, which requires a large set of PCSs to be reliable. In an alternative approach, we used multiple Gd3+-Gd3+ distances measured by double electron-electron resonance (DEER) experiments to define the metal position, allowing Δχ-tensor determinations from more robust 5-parameter fits that can be performed with a relatively sparse set of PCSs. Using this approach with the 32 kDa E. coli aspartate/glutamate binding protein (DEBP), we demonstrate a structural transition between substrate-bound and substrate-free DEBP, supported by PCSs generated by C3-Tm3+ and C3-Tb3+ tags attached to a genetically encoded p-azidophenylalanine residue. The significance of small PCSs was magnified by considering the difference between the chemical shifts measured with Tb3+ and Tm3+ rather than involving a diamagnetic reference. The integrative sparse data approach developed in this work makes poorly soluble proteins of limited stability amenable to structural studies in solution, without having to rely on cysteine mutations for tag attachment.",
keywords = "Double electron-electron resonance, E. coli aspartate/glutamate binding protein, Integrative structural biology, Lanthanide tag, Pseudocontact shift",
author = "Abdelkader, {Elwy H} and Xuejun Yao and Akiva Feintuch and Adams, {Luke A} and Luigi Aurelio and Bimbil Graham and Daniella Goldfarb and Gottfried Otting",
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Abdelkader, EH, Yao, X, Feintuch, A, Adams, LA, Aurelio, L, Graham, B, Goldfarb, D & Otting, G 2016, 'Pulse EPR-enabled interpretation of scarce pseudocontact shifts induced by lanthanide binding tags' Journal of Biomolecular NMR, vol. 64, no. 1, pp. 39-51. https://doi.org/10.1007/s10858-015-0003-z

Pulse EPR-enabled interpretation of scarce pseudocontact shifts induced by lanthanide binding tags. / Abdelkader, Elwy H; Yao, Xuejun; Feintuch, Akiva; Adams, Luke A; Aurelio, Luigi; Graham, Bimbil; Goldfarb, Daniella; Otting, Gottfried.

In: Journal of Biomolecular NMR, Vol. 64, No. 1, 2016, p. 39-51.

Research output: Contribution to journalArticleResearchpeer-review

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AU - Abdelkader, Elwy H

AU - Yao, Xuejun

AU - Feintuch, Akiva

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AU - Aurelio, Luigi

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AU - Goldfarb, Daniella

AU - Otting, Gottfried

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AB - Pseudocontact shifts (PCS) induced by tags loaded with paramagnetic lanthanide ions provide powerful long-range structure information, provided the location of the metal ion relative to the target protein is known. Usually, the metal position is determined by fitting the magnetic susceptibility anisotropy (Δχ) tensor to the 3D structure of the protein in an 8-parameter fit, which requires a large set of PCSs to be reliable. In an alternative approach, we used multiple Gd3+-Gd3+ distances measured by double electron-electron resonance (DEER) experiments to define the metal position, allowing Δχ-tensor determinations from more robust 5-parameter fits that can be performed with a relatively sparse set of PCSs. Using this approach with the 32 kDa E. coli aspartate/glutamate binding protein (DEBP), we demonstrate a structural transition between substrate-bound and substrate-free DEBP, supported by PCSs generated by C3-Tm3+ and C3-Tb3+ tags attached to a genetically encoded p-azidophenylalanine residue. The significance of small PCSs was magnified by considering the difference between the chemical shifts measured with Tb3+ and Tm3+ rather than involving a diamagnetic reference. The integrative sparse data approach developed in this work makes poorly soluble proteins of limited stability amenable to structural studies in solution, without having to rely on cysteine mutations for tag attachment.

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