Improved photo-CIDNP methods for studying protein structure and folding |
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Authors: | Kiminori Maeda Charles E Lyon Jakob J Lopez Masa Cemazar Christopher M Dobson PJ Hore |
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Institution: | (1) Oxford Centre for Molecular Sciences, UK;(2) Physical and Theoretical Chemistry Laboratory, UK;(3) New Chemistry Laboratory, Oxford University, Oxford, OX1 3QZ, U.K. |
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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- -lactalbumin, and by real-time CIDNP observation of the refolding of bovine apo- -lactalbumin following rapid dilution from a high concentration of chemical denaturant. |
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