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

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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.

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Acknowledgments

We thank Vincenzo Venditti and Mengli Cai for helpful suggestions, Dusty Baber and Jinfa Ying for assistance with NMR spectrometers, and Evan Brooks and Mária Balog for expert technical assistance. This work was in part supported by funds from the Intramural Program of the NIH, NIDDK and the Intramural AIDS Targeted Antiviral Program of the Office of the Director of the NIH (to G.M.C.), NIH grants R01EY05216 (to W.L.H.), and the Jules Stein Professorship Endowment (to W.L.H.). The synthesis of new spin label reagents was supported by Hungarian National Research Funds (OTKA K81123).

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Authors and Affiliations

  1. Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MA, 20892-0520, USA

    Nicolas L. Fawzi, Nicholas J. Anthis & G. Marius Clore

  2. Jules Stein Eye Institute and Department of Chemistry and Biochemistry, University of California, Los Angeles, CA, 90095, USA

    Mark R. Fleissner & Wayne L. Hubbell

  3. Institute of Organic and Medicinal Chemistry, University of Pécs, Szigeti str. 12, 7624, Pécs, Hungary

    Tamás Kálai & Kálmán Hideg

Authors
  1. Nicolas L. Fawzi
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  2. Mark R. Fleissner
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  3. Nicholas J. Anthis
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  4. Tamás Kálai
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  5. Kálmán Hideg
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  6. Wayne L. Hubbell
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  7. G. Marius Clore
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Correspondence to Wayne L. Hubbell or G. Marius Clore.

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Nicolas L. Fawzi, Mark R. Fleissner and Nicholas J. Anthis contributed equally.

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Fawzi, N.L., Fleissner, M.R., Anthis, N.J. et al. A rigid disulfide-linked nitroxide side chain simplifies the quantitative analysis of PRE data. J Biomol NMR 51, 105 (2011). https://doi.org/10.1007/s10858-011-9545-x

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