Conformational studies of a hyperthermostable enzyme.
The structural features of the hyperthermophilic endo-beta-1,3-glucanase from Pyrococcus furiosus were studied using circular dichroism, steady-state and time-resolved fluorescence spectroscopy and anisotropy. Upon heat and chemical treatment the folded and denatured states of the protein were characterized by distinguishable spectral profiles that identified a number of conformational states. The fluorescence methods showed that the spectral differences arose from changes in the local environment around specific tryptophan residues in the native, partially folded, partially unfolded and completely unfolded state. A structural resemblance was observed between the native protein and the structurally perturbed state which resulted after heat treatment at 110 degrees C. The enzyme underwent disruption of the native secondary and tertiary structure only after incubation at biologically extremely high temperatures (i.e. 150 degrees C), whilst in the presence of 8 m of guanidine hydrochloride the protein was partially unfolded.
Koutsopoulos
Sotirios
S
Laboratory of Physical Chemistry and Colloid Science, Wageningen University, the Netherlands. sotiris@mit.edu
van der Oost
John
J
Norde
Willem
W
D000817:Anilino Naphthalenesulfonates; D002942:Circular Dichroism; D004795:Enzyme Stability; D004693:Glucan Endo-1,3-beta-D-Glucosidase / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D020027:Pyrococcus furiosus / Q000201:enzymology*; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013997:Time Factors
D000817:Anilino Naphthalenesulfonates; C027132:1-anilino-8-naphthalenesulfonate; D004693:Glucan Endo-1,3-beta-D-Glucosidase