title: Improved photo-CIDNP methods for studying protein structure and folding.
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-alpha-lactalbumin, and by real-time CIDNP observation of the refolding of bovine apo-alpha-lactalbumin following rapid dilution from a high concentration of chemical denaturant.
authors.affiliation: Oxford Centre for Molecular Sciences, Physical and Theoretical Chemistry Laboratory, Oxford University, UK.
authors.forename: K
authors.initials: K
authors.lastname: Maeda
authors.forename: C E
authors.initials: CE
authors.lastname: Lyon
authors.forename: J J
authors.initials: JJ
authors.lastname: Lopez
authors.forename: M
authors.initials: M
authors.lastname: Cemazar
authors.forename: C M
authors.initials: CM
authors.lastname: Dobson
authors.forename: P J
authors.initials: PJ
authors.lastname: Hore
mesh_terms: D000818:Animals; D002417:Cattle; D005415:Flavins / Q000737:chemistry / Q000528:radiation effects; D006639:Histidine / Q000737:chemistry; D006861:Hydrogen Peroxide; D007768:Lactalbumin / Q000737:chemistry; D008027:Light; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D010084:Oxidation-Reduction; D017510:Protein Folding; D014364:Tryptophan / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry
chemical_list: D005415:Flavins; D014443:Tyrosine; D006639:Histidine; D014364:Tryptophan; D007768:Lactalbumin; D006861:Hydrogen Peroxide