In order to clarify the role played by the N-terminal region for the conformational stability of the thermophilic esterase 2 (EST2) from Alicyclobacillus acidocaldarius, two mutant forms have been investigated: a variant obtained by deleting the first 35 residues at the N-terminus (EST2-36del), and a variant obtained by mutating Lys102 to Gln (K102Q) to perturb the N-terminus by destroying the salt bridge E43-K102. The temperature- and denaturant-induced unfolding of EST2 and the two mutant forms have been studied by means of circular dichroism (CD), differential scanning calorimetry (DSC) and fluorescence measurements. In line with its thermophilic origin, the denaturation temperature of EST2 is high: Td = 91 °C and 86 °C if detected by recording the CD signal at 222 nm and 290 nm, respectively. This difference suggests that the thermal denaturation process, even though reversible, is more complex than a two-state N right arrow over left arrow D transition. The non-two-state behaviour is more pronounced in the case of the two mutant forms. The complex DSC profiles of EST2 and both mutant forms have been analysed by means of a deconvolution procedure. The thermodynamic parameters characterizing the two transitions obtained in the case of EST2 are: Td,1 = 81 °C, ΔdH1 = 440 kJ mol− 1, ΔdCp,1 = 7 kJ K− 1mol− 1, Td,2 = 86 °C, ΔdH2 = 710 kJ mol− 1, and ΔdCp,2 = 9 kJ K− 1mol− 1. The first transition occurs at lower temperatures in the two mutant forms, whereas the second transition is always centred at 86 °C. The results indicate that EST2 possesses two structural domains whose coupling is tight in the wild-type protein, but markedly weakens in the two mutant forms as a consequence of the perturbations in the N-terminal region.

Role of the N-terminal region for the conformational stability of esterase 2 from Alicyclobacillus acidocaldarius.

BARONE, GUIDO;ROSSI, MOSE';DEL VECCHIO, POMPEA GIUSEPPINA GRAZIA
2007

Abstract

In order to clarify the role played by the N-terminal region for the conformational stability of the thermophilic esterase 2 (EST2) from Alicyclobacillus acidocaldarius, two mutant forms have been investigated: a variant obtained by deleting the first 35 residues at the N-terminus (EST2-36del), and a variant obtained by mutating Lys102 to Gln (K102Q) to perturb the N-terminus by destroying the salt bridge E43-K102. The temperature- and denaturant-induced unfolding of EST2 and the two mutant forms have been studied by means of circular dichroism (CD), differential scanning calorimetry (DSC) and fluorescence measurements. In line with its thermophilic origin, the denaturation temperature of EST2 is high: Td = 91 °C and 86 °C if detected by recording the CD signal at 222 nm and 290 nm, respectively. This difference suggests that the thermal denaturation process, even though reversible, is more complex than a two-state N right arrow over left arrow D transition. The non-two-state behaviour is more pronounced in the case of the two mutant forms. The complex DSC profiles of EST2 and both mutant forms have been analysed by means of a deconvolution procedure. The thermodynamic parameters characterizing the two transitions obtained in the case of EST2 are: Td,1 = 81 °C, ΔdH1 = 440 kJ mol− 1, ΔdCp,1 = 7 kJ K− 1mol− 1, Td,2 = 86 °C, ΔdH2 = 710 kJ mol− 1, and ΔdCp,2 = 9 kJ K− 1mol− 1. The first transition occurs at lower temperatures in the two mutant forms, whereas the second transition is always centred at 86 °C. The results indicate that EST2 possesses two structural domains whose coupling is tight in the wild-type protein, but markedly weakens in the two mutant forms as a consequence of the perturbations in the N-terminal region.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11588/103195
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