Commitment to Folded and Aggregated States Occurs Late in Interleukin-1β Folding

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Abstract
A point mutation, lysine 97 to isoleucine, in the all-β cytokine interleukin-1β (IL-1β) 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 λ2 of 4.0 s-1, K97I commits to folding at a significantly slower rate λCF of 0.021 s-1. This rate of commitment to folding is in excellent agreement with the observed rate of K97I native state formation (λ1 = 0.018 s-1). K97I also commits slowly to aggregation at an observed rate λCA 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.