The Folding of Alpha-1-Proteinase Inhibitor: Kinetic vs Equilibrium Control

Abstract

Previous folding studies of α-1-proteinase inhibitor (α1-PI), which regulates the activity of the serine protease human neutrophil elastase, show an intermediate state at ∼1.5 M guanidine–HCl (Gu). For the normal form of α1-PI, we demonstrate the reversible formation of the same stable distribution of monomeric and polymeric intermediates after ∼1 h in 1.5 M Gu at ∼23°C from fully folded or fully unfolded α1-PI at similar final total concentrations and show that the stable distribution of monomeric and polymeric intermediates conforms with the law of mass action. We attribute these observations to an apparent equilibrium among intermediates. Our CD data are compatible with the intermediates having slightly relaxed structures relative to that of fully folded α1-PI and, thus, with the polymeric intermediates having a loop–sheet structure. Furthermore, we observe that the rates of folding (fast and slow terms) from the intermediate state are the same as those from the fully unfolded state, thereby supporting the contention that this intermediate state is on the folding pathway. We attribute the tendency of the Z mutant protein to polymerize/aggregate to an increased rate of the monomeric intermediate to form the apparent equilibrium distribution of intermediate species relative to its rate of folding to give intact α1-PI.