Aggregation events occur prior to stable intermediate formation during refolding of interleukin 1 beta

A point mutation, lysine 97 --> isoleucine (K97I), in a surface loop in the beta-sheet protein interleukin 1 beta (IL-1 beta), exhibits increased leve ls of inclusion body (IB) formation relative to the wildtype protein (WT) w hen expressed in Escherichia coli. Despite the common observation that less stable proteins are often found in IBs, K97I is more stable than WT. We ex amined the folding pathway of the mutant and wild-type proteins at pH 6.5 a nd 25 degrees C with-manual-mixing and stopped-flow optical spectroscopy to determine whether changes in the properties of transiently populated speci es 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 e vents (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 r eplacement affects the earlier steps in the refolding pathway. Aggregation, absent in the WT refolding reaction, occurs in K97I above a critical prote in concentration of 18 mu M. This observation is consistent with an initial nucleation step mediating protein aggregation. Stopped-flow kinetic studie s of the K97I aggregation process demonstrate that K97I aggregates most rap idly during the earliest refolding times, when unfolded protein conformers remain highly populated and the concentration of folding intermediates is l ow. Folding and aggregation studies together support a model in which the f ormation of stable folding intermediates afford protection against further K97I aggregation.