Calcium-induced tertiary structure modifications of endo-B-1,3-glucanase form Pyrococcus furiosus in 7.9 M guanidinium chloride
Chiaraluce, R. ; Gianese, G. ; Angelaccio, S. ; Florio, R. ; Lieshout, J.F.T. van; Oost, J. van der; Consalvi, V. - \ 2005
Biochemical Journal 386 (2005)3. - ISSN 0264-6021 - p. 515 - 524.
transform infrared-spectroscopy - protein secondary structure - 3-dimensional structures - ftir spectroscopy - free-energy - recognition - database - endo-beta-1,3-glucanase - intermediate - alignments
The family 16 endo-b-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-b-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
In situ structure and activity studies of an enzyme adsorbed on spectroscopically undetectable particles
Koutsopoulos, S. ; Tjeerdsma, A.M. ; Lieshout, J.F.T. van; Oost, J. van der; Norde, W. - \ 2005
Biomacromolecules 6 (2005)3. - ISSN 1525-7797 - p. 1176 - 1184.
archaeon pyrococcus-furiosus - solid-liquid interfaces - circular-dichroism - proteins - adsorption - endo-beta-1,3-glucanase - extremophiles - surfaces - features - sugar
The structural characteristics and the activity of a hyperthermophilic endoglucanase were investigated upon adsorption. Silica (hydrophilic) and Teflon (hydrophobic) surfaces were selected for the study. The materials were specially designed so that the interaction of the particles with light was negligible, and the enzyme conformation in the adsorbed state was monitored in situ. The adsorption isotherms were determined, and the adsorbed endoglucanase was studied using a number of spectroscopic techniques, enzymatic activity tests, and dynamic light scattering. Experiments were performed at pH values below, at, and above the isoelectric point of the enzyme. It was shown that the enzyme adsorbed on the hydrophobic surface of Teflon with higher affinity as compared to the hydrophilic silica nanoparticles. In all cases, adsorption was followed by (slight) changes in the secondary structure resulting in decreased -structural content. The changes were more profound upon adsorption on Teflon. The adsorbed enzyme remained active in the adsorbed state in spite of the structural changes induced when interacting with the surfaces