Thermodynamic and kinetic stability of a large multi-domain enzyme from the hyperthermophile Aeropyrum pernix
2010 (English)In: Extremophiles, ISSN 1431-0651, E-ISSN 1433-4909, Vol. 14, no 2, 213-223 p.Article in journal (Refereed) Published
The multi-domain enzyme isocitrate dehydrogenase from the hyperthermophile Aeropyrum pernix was studied by denaturant-induced unfolding. At pH 7.5, changes in circular dichroism ellipticity and intrinsic fluorescence showed a complex unfolding transition, whereas at pH 3.0, an apparently two-state and highly reversible unfolding occurred. Analytical ultracentrifugation revealed the dissociation from dimer to monomer at pH 3.0. The thermodynamic and kinetic stability were studied at pH 3.0 to explore the role of inter-domain interactions independently of inter-subunit interplay on the wild type and R211M, a mutant where a seven-membered inter-domain ionic network has been disrupted. The unfolding and folding transitions occurred at slightly different denaturant concentrations even after prolonged equilibration time. The difference between the folding and the unfolding profiles was decreased in the mutant R211M. The apparent Gibbs free energy decreased approximately 2 kcal/mol and the unfolding rate increased 4.3-fold in the mutant protein, corresponding to a decrease in activation free energy of unfolding of 0.86 kcal/mol. These results suggest that the inter-domain ionic network might be responsible for additional stabilization through a significant kinetic barrier in the unfolding pathway that could also explain the larger difference observed between the folding and unfolding transitions of the wild type.
Place, publisher, year, edition, pages
2010. Vol. 14, no 2, 213-223 p.
Hyperthermophiles, Protein stability, Thermodynamic stability, Kinetic stability, Ionic networks, Multi-domain protein, Isocitrate dehydrogenase
IdentifiersURN: urn:nbn:se:su:diva-50505DOI: 10.1007/s00792-009-0300-0ISI: 000275414800007OAI: oai:DiVA.org:su-50505DiVA: diva2:383541
authorCount :72011-01-052010-12-282011-01-05Bibliographically approved