title: Aggregation events occur prior to stable intermediate formation during refolding of interleukin 1beta.
abstract: A point mutation, lysine 97 --> isoleucine (K97I), in a surface loop in the beta-sheet protein interleukin 1beta (IL-1beta), exhibits increased levels of inclusion body (IB) formation relative to the wild-type protein (WT) when expressed in Escherichia coli. Despite the common observation that less stable proteins are often found in IBs, K97I is more stable than WT. We examined the folding pathway of the mutant and wild-type proteins at pH 6.5 and 25 degrees C with manual-mixing and stopped-flow optical spectroscopy to determine whether changes in the properties of transiently populated species in vitro correlate with the observation of increased aggregation in vivo. The refolding reactions of the WT and K97I proteins are both described by three exponential processes. Two exponential processes characterize fast events (0.1-1.0 s) in folding while the third exponential process correlates with a slow (70 s) single pathway to and from the native state. The K97I replacement affects the earlier steps in the refolding pathway. Aggregation, absent in the WT refolding reaction, occurs in K97I above a critical protein concentration of 18 microM. This observation is consistent with an initial nucleation step mediating protein aggregation. Stopped-flow kinetic studies of the K97I aggregation process demonstrate that K97I aggregates most rapidly during the earliest refolding times, when unfolded protein conformers remain highly populated and the concentration of folding intermediates is low. Folding and aggregation studies together support a model in which the formation of stable folding intermediates afford protection against further K97I aggregation.
authors.affiliation: Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California 92093-0359, USA.
authors.forename: J M
authors.initials: JM
authors.lastname: Finke
authors.forename: M
authors.initials: M
authors.lastname: Roy
authors.forename: B H
authors.initials: BH
authors.lastname: Zimm
authors.forename: P A
authors.initials: PA
authors.lastname: Jennings
mesh_terms: D019943:Amino Acid Substitution; D003001:Cloning, Molecular; D004355:Drug Stability; D004926:Escherichia coli; D019791:Guanidine; D007375:Interleukin-1 / Q000737:chemistry* / Q000378:metabolism*; D007532:Isoleucine; D007700:Kinetics; D008239:Lysine; D046911:Macromolecular Substances; D008958:Models, Molecular; D017354:Point Mutation; D011489:Protein Denaturation; D017510:Protein Folding; D020673:Protein Renaturation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012542:Scattering, Radiation
chemical_list: D007375:Interleukin-1; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D007532:Isoleucine; D019791:Guanidine; D008239:Lysine