TAILIEUCHUNG - Báo cáo khoa học: Kinetically controlled refolding of a heat-denatured hyperthermostable protein

The thermal denaturation of endo-b-1,3-glucanase from the hyperthermo-philic microorganismPyrococcus furiosuswas studied by calorimetry. The calorimetric profile revealed two transitions at 109 and 144 C, correspond-ing to protein denaturation and complete unfolding, respectively, as shown by circular dichroism and fluorescence spectroscopy data. | ỊFEBS Journal Kinetically controlled refolding of a heat-denatured hyperthermostable protein Sotirios Koutsopoulos1 John van der Oost2 and Willem Norde1 3 1 Laboratory of PhysicalChemistry and Colloid Science Wageningen University the Netherlands 2 Laboratory of Microbiology Wageningen University the Netherlands 3 Department of BiomedicalEngineering University of Groningen the Netherlands Keywords calorimetry endo-b-1 3-glucanase hyperthermostable enzyme protein refolding Correspondence S. Koutsopoulos Center for Biomedical Engineering Massachusetts Institute of Technology NE47-Room 307 500 Technology Square Cambridge MA 02139-4307 USA Fax 31 617 258 5239 Tel 31 617 324 7612 E-mail sotiris@ The thermal denaturation of endo-b-1 3-glucanase from the hyperthermo-philic microorganism Pyrococcus furiosus was studied by calorimetry. The calorimetric profile revealed two transitions at 109 and 144 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. Received 30 July 2007 accepted 21 September 2007 doi Hyperthermophilic microorganisms which often belong to the Archaea are able to grow optimally at 100 C or higher 1 . After their discovery it was necessary to revise our ideas about the mechanisms involved in the maintenance of protein structural integrity and function at elevated temperatures 2 3 . For the stabilization of proteins at high temperatures a concerted optimization of structural features is employed. These include reduced solvent-exposed surface area 4 increased packing .

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