title: Proton NMR of aequorin. Structural changes concomitant with calcium-independent light emission.
abstract: Aequorin, a Ca(II)-sensitive bioluminescent protein from jellyfish, emits light at 469 nm from an excited state of a substituted pyrazine (oxyluciferin) which results from the oxidation of a chromophore molecule that is noncovalently bound to the protein. The chromophore is oxidized when Ca(II) or other activating metal ions are bound by aequorin. In the absence of Ca(II), spontaneous emission of light, referred to as Ca(II)-independent light emission, occurs at a rate less than 10(-6) of that for Ca(II)-induced emission. Proton nuclear magnetic resonance (NMR), circular dichroism (CD), and fluorescence were used to study structural changes of aequorin accompanying Ca(II)-independent light emission. Time course studies by 1H NMR and CD demonstrate that as a result of Ca(II)-independent light emission, aequorin progressively changes from a rigid, fully active form showing little segmental mobility to a practically unfolded, discharged (i.e., inactive) form in which a number of amino acid residues are significantly mobile. This slow discharged protein (SDP) is distinct in nature and conformation from aequorin which has been discharged by Ca(II), i.e., the blue fluorescent protein. The rate of Ca(II)-independent discharge of aequorin is substantially reduced in the presence of excess Mg(II); the time constant for inactivation at 5 degrees C is 30 days with no Mg(II) present and 70 days with Mg(II) present. The NMR spectra are nearly identical at a given stage of inactivation whether or not Mg(II) is present. Oxyluciferin remains bound to SDP. If it is removed, however, by column chromatography, the resulting apo-SDP partially refolds, and the segmental mobility acquired in the formation of SDP is significantly attenuated particularly for some of the aromatic amino acid residues.
mesh_terms: D000331:Aequorin / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002942:Circular Dichroism; D049454:Firefly Luciferin / Q000031:analogs & derivatives / Q000302:isolation & purification; D007211:Indoles; D008027:Light; D008164:Luminescent Proteins / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D011487:Protein Conformation; D011719:Pyrazines; D037401:Scyphozoa / Q000378:metabolism; D013050:Spectrometry, Fluorescence
chemical_list: D007211:Indoles; D008164:Luminescent Proteins; D011719:Pyrazines; C014730:oxyluciferin; D000331:Aequorin; D049454:Firefly Luciferin; D002118:Calcium