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 interle ukin-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 t he aggregation reaction and determine when intervention may allow rescue of protein from aggregation during renaturation, we developed a novel applica tion of mass spectrometry using isotopic labeling to determine the step(s) at which K97I commits to either the native or aggregated state. Interesting ly, despite the early formation of a folding intermediate ensemble at an ob served rate lambda (2) of 4.0 s(-1), K97I commits to folding at a significa ntly slower rate lambda (CF) of 0.021 s(-1). This rate of commitment to fol ding is in excellent agreement with the observed rate of K97I native state formation (lambda (1) = 0.018 s(-1)). K97I also commits slowly to aggregati on 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 fold ing and aggregation processes are consistent with a nucleation-dependent mo del of aggregation.