Calcium-induced tertiary structure modifications of endo-beta-1,3-glucanase from Pyrococcus furiosus in 7.9 M guanidinium chloride.
The family 16 endo-beta-1,3 glucanase from the extremophilic archaeon Pyrococcus furiosus is a laminarinase, which in 7.9 M GdmCl (guanidinium chloride) maintains a significant amount of tertiary structure without any change of secondary structure. The addition of calcium to the enzyme in 7.9 M GdmCl causes significant changes to the near-UV CD and fluorescence spectra, suggesting a notable increase in the tertiary structure which leads to a state comparable, but not identical, to the native state. The capability to interact with calcium in 7.9 M GdmCl with a consistent recovery of native tertiary structure is a unique property of this extremely stable endo-beta-1,3 glucanase. The effect of calcium on the thermodynamic parameters relative to the GdmCl-induced equilibrium unfolding has been analysed by CD and fluorescence spectroscopy. The interaction of calcium with the native form of the enzyme is studied by Fourier-transform infrared spectroscopy in the absorption region of carboxylate groups and by titration in the presence of a chromophoric chelator. A homology-based model of the enzyme is generated and used to predict the putative binding site(s) for calcium and the structural interactions potentially responsible for the unusual stability of this protein, in comparison with other family 16 glycoside hydrolases.
Dipartimento di Scienze Biochimiche A Rossi Fanelli, Università La Sapienza, P.le A. Moro 5, 00185 Rome, Italy.
Roberta
R
Chiaraluce
Giulio
G
Gianese
Sebastiana
S
Angelaccio
Rita
R
Florio
Johan F T
JF
van Lieshout
John
J
van der Oost
Valerio
V
Consalvi
D000595:Amino Acid Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology*; D002942:Circular Dichroism; D004693:Glucan Endo-1,3-beta-D-Glucosidase / Q000737:chemistry* / Q000378:metabolism*; D019791:Guanidine / Q000494:pharmacology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding; D017434:Protein Structure, Tertiary / Q000187:drug effects; D020027:Pyrococcus furiosus / Q000201:enzymology*; D016415:Sequence Alignment; D013050:Spectrometry, Fluorescence; D017550:Spectroscopy, Fourier Transform Infrared; D013816:Thermodynamics
D004693:Glucan Endo-1,3-beta-D-Glucosidase; D019791:Guanidine; D002118:Calcium