title: Thermodynamic and kinetic stability of a large multi-domain enzyme from the hyperthermophile Aeropyrum pernix.
abstract: 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.
authors.affiliation: Karolinska Institutet, Center of Bioscience, 141 57 Huddinge, Sweden. mikael.karlstrom@dbb.su.se
authors.forename: Mikael
authors.initials: M
authors.lastname: Karlström
authors.forename: Roberta
authors.initials: R
authors.lastname: Chiaraluce
authors.forename: Laura
authors.initials: L
authors.lastname: Giangiacomo
authors.forename: Ida Helene
authors.initials: IH
authors.lastname: Steen
authors.forename: Nils-Kåre
authors.initials: NK
authors.lastname: Birkeland
authors.forename: Rudolf
authors.initials: R
authors.lastname: Ladenstein
authors.forename: Valerio
authors.initials: V
authors.lastname: Consalvi
mesh_terms: D041522:Aeropyrum / Q000201:enzymology* / Q000235:genetics; D019943:Amino Acid Substitution; D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007521:Isocitrate Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D017510:Protein Folding; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013816:Thermodynamics; D056811:Unfolded Protein Response; D014508:Urea
chemical_list: D019843:Archaeal Proteins; D011994:Recombinant Proteins; D014508:Urea; D007521:Isocitrate Dehydrogenase