Photochemically-induced dynamic nuclear polarization proton-NMR of aequorin discharged by calcium-independent light emission.

Abstract

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 alpha 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.