title: Folding of firefly (Photinus pyralis) luciferase: aggregation and reactivation of unfolding intermediates.
abstract: The guanidine-induced unfolding of firefly (Photinus pyralis) luciferase involves two inactive equilibrium intermediates and is freely reversible at low protein concentration and low temperature. However, reactivation is exceedingly slow so that the equilibrium is attained only after several days of incubation and reactivation yields decrease strongly with increasing protein concentration, suggesting that aggregation is a competing side reaction [Herbst et al. (1997) J. Biol. Chem. 272, 7099-7105]. We investigated the role of the equilibrium intermediates in the aggregation process using size-exclusion chromatography and dynamic light scattering to monitor their association state. Although the more unfolded intermediate aggregated much more rapidly, both intermediates associated irreversibly without a conformational change visible by fluorescence or circular dichroism, forming small oligomers which remained soluble in the presence of the denaturant. The association kinetics are compatible with a nucleated polymerization mechanism. Unfolding kinetics at 1 M denaturant indicated the presence of a further inactive intermediate capable to reactivate rapidly with kinetics similar to those observed for luciferase reactivation in the presence of cell extracts. The data suggest a kinetic trap in luciferase refolding that is accessible from both equilibrium intermediate conformations and is avoided in the presence of molecular chaperones.
authors.affiliation: Universität Regensburg, Institut für Biophysik und Physikalische Biochemie, D-93040 Regensburg, Germany.
authors.forename: R
authors.initials: R
authors.lastname: Herbst
authors.forename: K
authors.initials: K
authors.lastname: Gast
authors.forename: R
authors.initials: R
authors.lastname: Seckler
mesh_terms: D000818:Animals; D001517:Coleoptera; D008156:Luciferases / Q000737:chemistry* / Q000378:metabolism; D018832:Molecular Chaperones / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding
chemical_list: D018832:Molecular Chaperones; D008156:Luciferases