Early aggregated States in the folding of interleukin-1ß.

Kinetic data measured from folding of the protein interleukin-1ß fits best to three exponential phases when studied with tryptophan fluorescence but only two exponential phases when measured using other methods. The technique of ANS fluorescence was used to determine whether the additional phase observed in tryptophan fluorescence was also detected with ANS dye binding. Unlike trytophan fluorescence, the ANS fluorescence was highly dependent on the concentration of protein present during the folding experiment. Experimental controls provide evidence that ANS binds to protein aggregates, present at higher concentrations and absent at lower concentrations. Protein concentration-dependent folding studies demonstrate that, at lower interleukin-1ß concentrations, tryptophan fluorescence kinetics can be fit adequately with a two exponential fit. This study indicates that (1) measured interleukin-1ß folding kinetics fit to a 2 phase model and (2) at higher protein concentrations, transient association of IL-1ß may result in a kinetic fit of 3 phases.

Commitment to folded and aggregated states occurs late in interleukin-1 beta folding.

A point mutation, lysine 97 to isoleucine, in the all-beta cytokine interleukin-1 beta (IL-1 beta) exhibits an increased propensity to form inclusion bodies in vivo and aggregates in vitro. In an effort to better understand the aggregation reaction and determine when intervention may allow rescue of protein from aggregation during renaturation, we developed a novel application of mass spectrometry using isotopic labeling to determine the step(s) at which K97I commits to either the native or aggregated state. Interestingly, despite the early formation of a folding intermediate ensemble at an observed rate lambda(2) of 4.0 s(-1), K97I commits to folding at a significantly slower rate lambda(CF) of 0.021 s(-1). This rate of commitment to folding is in excellent agreement with the observed rate of K97I native state formation (lambda(1) = 0.018 s(-1)). K97I also commits slowly to aggregation at an observed rate lambda(CA) of 0.023 s(-1). Earlier folding species and aggregates present prior to these commitment steps are likely to be in a reversible equilibrium between monomeric folding intermediates and higher-order oligomers. Kinetic and equilibrium experimental measurements of folding and aggregation processes are consistent with a nucleation-dependent model of aggregation.

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

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.