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Improved photo-CIDNP methods for studying protein structure and folding

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Abstract

Two new techniques offering considerable improvements in the quality of 1H photo-CIDNP spectra of proteins are demonstrated. Both focus on the problem of progressive photo-degradation of the flavin dye used to generate polarization in exposed tryptophan, tyrosine and histidine side-chains. One approach uses rapid addition and removal of protein/flavin solution between light flashes to mix the NMR sample and introduce fresh dye into the laser-irradiated region. The other involves chemical oxidation of photo-reduced flavin by the addition of hydrogen peroxide. In both cases a larger number of scans can be accumulated before the flavin is exhausted than would otherwise be possible. The techniques are demonstrated by 600 MHz CIDNP-NOESY spectroscopy of bovine holo-α-lactalbumin, and by real-time CIDNP observation of the refolding of bovine apo-α-lactalbumin following rapid dilution from a high concentration of chemical denaturant.

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Authors and Affiliations

  1. Oxford Centre for Molecular Sciences, UK

    Kiminori Maeda, Charles E. Lyon, Jakob J. Lopez, Masa Cemazar, Christopher M. Dobson & P.J. Hore

  2. Physical and Theoretical Chemistry Laboratory, UK

    Kiminori Maeda, Charles E. Lyon, Jakob J. Lopez, Masa Cemazar & P.J. Hore

  3. New Chemistry Laboratory, Oxford University, Oxford, OX1 3QZ, U.K.

    Christopher M. Dobson

Authors
  1. Kiminori Maeda
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  2. Charles E. Lyon
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  3. Jakob J. Lopez
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  4. Masa Cemazar
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  5. Christopher M. Dobson
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  6. P.J. Hore
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Maeda, K., Lyon, C.E., Lopez, J.J. et al. Improved photo-CIDNP methods for studying protein structure and folding. J Biomol NMR 16, 235–244 (2000). https://doi.org/10.1023/A:1008351128089

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