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

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


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.

• Content Type Journal Article
• Category Article
• Pages 105-114
• DOI 10.1007/s10858-011-9545-x
• Authors
  • Nicolas L. Fawzi, Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MA 20892-0520, USA
  • Mark R. Fleissner, Jules Stein Eye Institute and Department of Chemistry and Biochemistry, University of California, Los Angeles, CA 90095, USA
  • Nicholas J. Anthis, Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MA 20892-0520, USA
  • Tamás Kálai, Institute of Organic and Medicinal Chemistry, University of Pécs, Szigeti str. 12, 7624 Pécs, Hungary
  • Kálmán Hideg, Institute of Organic and Medicinal Chemistry, University of Pécs, Szigeti str. 12, 7624 Pécs, Hungary
  • Wayne L. Hubbell, Jules Stein Eye Institute and Department of Chemistry and Biochemistry, University of California, Los Angeles, CA 90095, USA
  • G. Marius Clore, Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MA 20892-0520, USA

• • Journal Journal of Biomolecular NMR
  • Online ISSN 1573-5001
  • Print ISSN 0925-2738
  • Journal Volume Volume 51
  • Journal Issue Volume 51, Numbers 1-2

Source: Journal of Biomolecular NMR