title: Collapsed intermediates in the reconstitution of dimeric aspartate aminotransferase from Escherichia coli.
abstract: Aspartate aminotransferase from Escherichia coli, which had been denatured by guanidinium chloride, refolded and reassembled to active dimers in two distinct phases. The unfolded monomer U collapsed within 20 s to an intermediate I* that was inactive, fluoresced more strongly than, but had the same peptide CD signal as the native dimer. The formation of crosslinkable dimers, as well as the recovery of enzyme activity, occurred with a biphasic progress curve which was independent of protein concentration. The half-lives of the two phases were 100 s and 2000 s. The data are consistent with a three-step mechanism, in which the overall rate of reassembly is determined by an isomerization of I* to the assembly-competent monomer M. The latter does not accumulate because it dimerizes rapidly to the active enzyme (D). Reassembly of the enzyme from the compact intermediate M*, which is stable at 1.0 M guanidinium chloride, also proceeded in a rapid and a slow phase. Moreover, the formation of M* from the unfolded state was rapid, whereas its refolding to the native dimer was slow. Both the transient intermediate I* and the equilibrium intermediate M* qualify as 'collapsed intermediate' or 'molten globule' states.
authors.affiliation: Department of Biophysical Chemistry, University of Basel, Switzerland.
authors.forename: B
authors.initials: B
authors.lastname: Leistler
authors.forename: M
authors.initials: M
authors.lastname: Herold
authors.forename: K
authors.initials: K
authors.lastname: Kirschner
mesh_terms: D001219:Aspartate Aminotransferases / Q000737:chemistry* / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D019791:Guanidine; D006146:Guanidines; D007700:Kinetics; D046911:Macromolecular Substances; D008962:Models, Theoretical; D011487:Protein Conformation; D011489:Protein Denaturation; D013050:Spectrometry, Fluorescence
chemical_list: D003432:Cross-Linking Reagents; D006146:Guanidines; D046911:Macromolecular Substances; D001219:Aspartate Aminotransferases; D019791:Guanidine