Naslov
Thermal unfolding of eosinophil cationic protein/ribonuclease 3: A nonreversible process

Autori
Nikolovski, Zoran ; Buzón, Víctor ; Ribó, Marc ; Moussaoui, Mohammed ; Vilanova, Maria ; Cuchillo, Claudi M. ; Cladera, Josep ; Nogués, M. Victòria

Izvornik
Protein science (0961-8368) 15 (2006), 12; 2816-2827

Vrsta, podvrsta i kategorija rada
Radovi u časopisima, članak, znanstveni

Ključne riječi
eosinophil cationic protein ; ribonucleases ; thermal stability ; protein folding

Sažetak
Eosinophil cationic protein (ECP)/ribonuclease 3 is a member of the RNase A superfamily involved in inflammatory processes mediated by eosinophils. ECP is bactericidal, helminthotoxic, and cytotoxic to tracheal epithelium cells and to several mammalian cell lines although its RNase activity is low. We studied the thermal stability of ECP by fourth-derivative UV absorbance spectra, circular dichroism, differential scanning calorimetry, and Fourier transform infrared spectroscopy. The T1/2 values obtained with the different techniques were in very good agreement (T1/2
72°C), and the stability was maintained in the pH range between 5 and 7. The ECP calorimetric melting curve showed, in addition to the main transition, a pretransitional conformational change with a T1/2 of 44°C. Both calorimetric transitions disappeared after successive re-heatings, and the ratio DH versus DHvH of 2.2 indicated a significant deviation from the two-state model. It was observed that the thermal unfolding was irreversible. The unfolding process gives rise to changes in the environment of aromatic amino acids that are partially maintained in the refolded protein with the loss of secondary structure and the formation of oligomers. From the thermodynamic analysis of ECP variants, the contribution of specific amino acids, such as Trp10 and the region 115–122, to thermal stability was also determined. The high thermal stability of ECP may contribute to its resistance to degradation when the protein is secreted to the extracellular medium during the immune response.

Izvorni jezik
Engleski

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