Role of the N-terminal region for the conformational stability of esterase 2 from Alicyclobacillus acidocaldarius.
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: T(d)=91 degrees C and 86 degrees 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 Nright arrow over left arrowD 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: T(d,1)=81 degrees C, Delta(d)H(1)=440 kJ mol(-1), Delta(d)C(p,1)=7 kJ K(-1)mol(-1), T(d,2)=86 degrees C, Delta(d)H(2)=710 kJ mol(-1), and Delta(d)C(p,2)=9 kJ K(-1)mol(-1). The first transition occurs at lower temperatures in the two mutant forms, whereas the second transition is always centred at 86 degrees 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.
Foglia
Fabrizia
F
Dipartimento di Chimica, Università di Napoli Federico II, Via Cintia, 80126 Napoli, Italy.
Mandrich
Luigi
L
Pezzullo
Margherita
M
Graziano
Giuseppe
G
Barone
Guido
G
Rossi
Mosè
M
Manco
Giuseppe
G
Del Vecchio
Pompea
P
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D004795:Enzyme Stability; D004950:Esterases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D016937:Gram-Positive Endospore-Forming Rods / Q000201:enzymology*; D009154:Mutation; D011487:Protein Conformation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D013056:Spectrophotometry, Ultraviolet; D013816:Thermodynamics
D001426:Bacterial Proteins; D004950:Esterases