The thermophilic esterase from Archaeoglobus fulgidus: structure and conformational dynamics at high temperature.
The esterase from the hyperthermophilic archaeon Archaeoglobus fulgidus is a monomeric protein with a molecular weight of about 35.5 kDa. The enzyme is barely active at room temperature, displaying the maximal enzyme activity at about 80 degrees C. We have investigated the effect of the temperature on the protein structure by Fourier-transform infrared spectroscopy. The data show that between 20 degrees C and 60 degrees C a small but significant decrease of the beta-sheet bands occurred, indicating a partial loss of beta-sheets. This finding may be surprising for a thermophilic protein and suggests the presence of a temperature-sensitive beta-sheet. The increase in temperature from 60 degrees C to 98 degrees C induced a decrease of alpha-helix and beta-sheet bands which, however, are still easily detected at 98 degrees C indicating that at this temperature some secondary structure elements of the protein remain intact. The conformational dynamics of the esterase were investigated by frequency-domain fluorometry and anisotropy decays. The fluorescence studies showed that the intrinsic tryptophanyl fluorescence of the protein was well represented by the three-exponential model, and that the temperature affected the protein conformational dynamics. Remarkably, the tryptophanyl fluorescence emission reveals that the indolic residues remained shielded from the solvent up to 80 degrees C, as shown from the emission spectra and by acrylamide quenching experiments. The relationship between enzyme activity and protein structure is discussed.
D'Auria
S
S
University of Maryland School of Medicine, Department of Biochemistry and Molecular Biology, Baltimore, USA.
Herman
P
P
Lakowicz
J R
JR
Bertoli
E
E
Tanfani
F
F
Rossi
M
M
Manco
G
G
D019611:Archaeoglobus fulgidus / Q000737:chemistry*; D003903:Deuterium; D004950:Esterases / Q000737:chemistry*; D006358:Hot Temperature; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence; D017550:Spectroscopy, Fourier Transform Infrared
D003903:Deuterium; D004950:Esterases