Human ceruloplasmin (CP) is a multicopper oxidase essential for normal iron homeostasis. The protein has six domains with one type-1 copper in each of domains 2, 4, and 6; the remaining coppers form a catalytic trinuclear cluster at the interface between domains 1 and 6. To assess the role of the coppers in CP thermal stability, we have probed the thermal unfolding process as a function of scan rate of holo- and apo-forms using several detection methods (circular dichroism, aromatic and 8-anilino-naphthalene-1-sulfonic acid fluorescence, visible absorption, activity, and differential scanning calorimetry). Both species of CP undergo irreversible thermal reactions to denatured states with significant residual structure. For identical scan rates, the thermal midpoint appears at temperatures 15-20 degrees higher for the holo- as compared with the apo- form. The thermal data for both forms were fit by a mechanistic model involving two consecutive, irreversible steps (N --> I --> D). The holo-intermediate, I, has lost one oxidized type-1 copper and secondary structure in at least one domain; however, the trinuclear copper cluster remains intact as it is functional in oxidase activity. The activation parameters obtained from the fits to the thermal transitions were used to assess the kinetic stability of apo- and holo-CP at physiological temperatures (i.e., at 37 degrees C). It emerges that native CP (i.e., with six coppers) is rather unstable and converts to I in <1 day at 37 degrees C. Nonetheless, this form remains intact for more than 2 weeks and may thus be a biologically relevant state of CP in vivo. In contrast, apo-CP unfolds rapidly: the denatured state is reached in <2 days at 37 degrees C. Study holds ProTherm entries: 23879, 23880, 23881, 23882, 23883, 23884, 23885, 23886, 23887, 23888, 23889, 23890, 23891, 23892, 23893, 23894, 23895, 23896, 23897, 23898, 23899, 23900, 23901, 23902, 23903, 23904, 23905, 23906, 23907, 23908 Extra Details: apo form; transition 1; scanning rate: 0.5 K/min copper; thermal unfolding; intermediate; kinetic stability
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:54 p.m.
|Number of data points||30|
|Proteins||Ceruloplasmin ; CERULOPLASMIN|
|Assays/Quantities/Protocols||Experimental Assay: Tm prot_conc:0.3-0.4 microM ; Experimental Assay: Tm prot_conc:3 microM ; Experimental Assay: Tm prot_conc:3-4 microM|