Photochemically‐induced dynamic nuclear polarization was used to identify exposed amino‐acid residues and to assign resonances in the 1H‐NMR spectrum of Ca(II)‐independent discharged (inactivated) aequorin. A previous nuclear magnetic resonance, circular dichroism and fluorescence study [Ray, B. D., Ho, S., Kemple, M. D., Prendergast, F. G. and Nageswara Rao, B. D. (1985) Biochemistry 24, 4280–4287] indicated that as the Ca(II)‐activated bioluminescent protein aequorin from jellyfish spontaneously emits light in the absence of Ca(II), it changes from a rigid, fully active form to a discharged form in which a number of amino‐acid residues are more mobile than in the native protein. Laser‐photochemically‐induced dynamic nuclear polarization experiments identified tryptophan and tyrosine residues, but not histidine residues, in Ca(II)‐independent discharged aequorin to be accessible to the flavin dye used. These exposed residues are also among the mobile residues of the Ca(II)‐independent discharged protein. Resonances of all the protons (including the α protons) of the accessible tryptophan and tyrosine residues were assigned with the aid of two‐dimensional photochemically‐induced dynamic nuclear polarization J‐correlated spectroscopy. The oxidized chromophore, from which light is emitted in aequorin, was not accessible to the dye in the Ca(II)‐independent discharged protein. No exposed residue was detected in the photochemically‐induced dynamic nuclear polarization spectrum of Ca(II)‐independent discharged aequorin from which the oxidized chromophore was removed, corroborating the previous finding that in this apo‐discharged form the protein partially refolds and thereby loses some of the mobility acquired in the formation of the Ca(II)‐independent discharged protein.
|Original language||English (US)|
|Number of pages||5|
|Journal||European Journal of Biochemistry|
|State||Published - Jan 1989|
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