A rigid disulfide-linked nitroxide side chain simplifies the quantitative analysis of PRE data

Nicolas L. Fawzi, Mark R. Fleissner, Nicholas J. Anthis, Tamás Kálai, Kálmán Hideg, Wayne L. Hubbell, G. Marius Clore

Research output: Contribution to journalArticle

44 Citations (Scopus)

Abstract

The measurement of 1H transverse paramagnetic relaxation enhancement (PRE) has been used in biomolecular systems to determine long-range distance restraints and to visualize sparsely-populated transient states. The intrinsic flexibility of most nitroxide and metal-chelating paramagnetic spin-labels, however, complicates the quantitative interpretation of PREs due to delocalization of the paramagnetic center. Here, we present a novel, disulfide-linked nitroxide spin label, R1p, as an alternative to these flexible labels for PRE studies. When introduced at solvent-exposed α-helical positions in two model proteins, calmodulin (CaM) and T4 lysozyme (T4L), EPR measurements show that the R1p side chain exhibits dramatically reduced internal motion compared to the commonly used R1 spin label (generated by reacting cysteine with the spin labeling compound often referred to as MTSL). Further, only a single nitroxide position is necessary to account for the PREs arising from CaM S17R1p, while an ensemble comprising multiple conformations is necessary for those observed for CaM S17R1. Together, these observations suggest that the nitroxide adopts a single, fixed position when R1p is placed at solvent-exposed α-helical positions, greatly simplifying the interpretation of PRE data by removing the need to account for the intrinsic flexibility of the spin label.

Original languageEnglish
Pages (from-to)105-114
Number of pages10
JournalJournal of Biomolecular NMR
Volume51
Issue number1-2
DOIs
Publication statusPublished - Sep 2011

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Spin Labels
Disulfides
Calmodulin
Chemical analysis
Muramidase
Chelation
Labeling
Cysteine
Paramagnetic resonance
Conformations
Labels
Metals
Proteins

Keywords

  • Electron paramagnetic resonance
  • Paramagnetic relaxation enhancement
  • Site-directed spin-labeling

ASJC Scopus subject areas

  • Spectroscopy
  • Biochemistry

Cite this

A rigid disulfide-linked nitroxide side chain simplifies the quantitative analysis of PRE data. / Fawzi, Nicolas L.; Fleissner, Mark R.; Anthis, Nicholas J.; Kálai, Tamás; Hideg, Kálmán; Hubbell, Wayne L.; Clore, G. Marius.

In: Journal of Biomolecular NMR, Vol. 51, No. 1-2, 09.2011, p. 105-114.

Research output: Contribution to journalArticle

Fawzi, Nicolas L. ; Fleissner, Mark R. ; Anthis, Nicholas J. ; Kálai, Tamás ; Hideg, Kálmán ; Hubbell, Wayne L. ; Clore, G. Marius. / A rigid disulfide-linked nitroxide side chain simplifies the quantitative analysis of PRE data. In: Journal of Biomolecular NMR. 2011 ; Vol. 51, No. 1-2. pp. 105-114.
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