title: The beta-glycosidase from the hyperthermophilic archaeon Sulfolobus solfataricus: enzyme activity and conformational dynamics at temperatures above 100 degrees C.
abstract: Enzymes from thermophilic organisms are stable and active at temperatures which rapidly denature mesophilic proteins. However, there is not yet a complete understanding of the structural basis of their thermostability and thermoactivity since for each protein there seems to exist special networks of interactions that make it stable under the desired conditions. Here we have investigated the activity and conformational dynamics above 100 degrees C of the beta-glycosidase isolated from the hyperthermophilic archaeon Sulfolobus solfataricus. This has been made possible using a special stainless steel optical pressure cell which allowed us to perform enzyme assays and fluorescence measurements up to 160 degrees C without boiling the sample. The beta-glycosidase from S. solfataricus showed maximal activity at 125 degrees C. The time-resolved fluorescence studies showed that the intrinsic tryptophanyl fluorescence emission of the protein was represented by a bimodal distribution with Lorential shape and that temperature strongly affected the protein conformational dynamics. Remarkably, the tryptophan emission reveals that the indolic residues remain shielded from the solvent even at 125 degrees C, as shown by shielding from quenching and restricted tryptophan solubility. The relationship between enzyme activity and protein structural dynamics is discussed.
authors.affiliation: Institute of Protein Biochemistry and Enzymology, C.N.R., Napoli, Italy.
authors.forename: S
authors.initials: S
authors.lastname: D'Auria
authors.forename: R
authors.initials: R
authors.lastname: Nucci
authors.forename: M
authors.initials: M
authors.lastname: Rossi
authors.forename: I
authors.initials: I
authors.lastname: Gryczynski
authors.forename: Z
authors.initials: Z
authors.lastname: Gryczynski
authors.forename: J R
authors.initials: JR
authors.lastname: Lakowicz
mesh_terms: D000465:Algorithms; D006358:Hot Temperature; D007202:Indicators and Reagents; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D016944:Sulfolobus / Q000201:enzymology*; D001617:beta-Glucosidase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D007202:Indicators and Reagents; D001617:beta-Glucosidase