Folding pathway of human α₁-antitrypsin: characterization of an intermediate that is active but prone to aggregation

Abstract

The folding-unfolding kinetics of human α1-antitrypsin ((α1-AT) were examined by monitoring intrinsic Trp fluorescence and extrinsic ANS fluorescence. While the unfolding of α1-AT followed a single exponential phase, refolding exhibited three exponential phases. The fast phase (T(1,r),< 40 sec), which was independent of urea concentration, appears to be hydrophobic collapse that may be limited by cis-trans isomerization of prolyl residue. The medium phase (T(2,r) = 200 sec) yielded an intermediate (I(N)),which is capable of elastase binding. The slowest (T(3,r), = 1000 sec) phase completes refolding to the native protein, which intersects with the unfolding kinetics at the same urea concentration (1.9 M) as the equilibrium midpoint Concentration-dependence of the amplitude of major refolding phases indicated that IN is prone to kinetic competition between the on-pathway to native protein and aggregation.