Kinetically controlled refolding of a heat-denatured hyperthermostable protein.
The thermal denaturation of endo-beta-1,3-glucanase from the hyperthermophilic microorganism Pyrococcus furiosus was studied by calorimetry. The calorimetric profile revealed two transitions at 109 and 144 degrees C, corresponding to protein denaturation and complete unfolding, respectively, as shown by circular dichroism and fluorescence spectroscopy data. Calorimetric studies also showed that the denatured state did not refold to the native state unless the cooling temperature rate was very slow. Furthermore, previously denatured protein samples gave well-resolved denaturation transition peaks and showed enzymatic activity after 3 and 9 months of storage, indicating slow refolding to the native conformation over time.
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
D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D006358:Hot Temperature; D007700:Kinetics; D017510:Protein Folding; D020027:Pyrococcus furiosus / Q000201:enzymology; D013050:Spectrometry, Fluorescence