Kinetic Instability of the Serpin Z α1-Antitrypsin Promotes Aggregation

The serpinopathies encompass a large number of diseases caused by inappropriate conformational change and self-association (polymerization) of a serpin (serine proteinase inhibitor) molecule. The most common serpinopathy is α1-antitrypsin (α1AT) deficiency, which is associated with an increased risk for liver cirrhosis, hepatocellular carcinoma and early-onset emphysema. The Z variant of α1AT, which accounts for 95% of all cases of α1AT deficiency, polymerizes during synthesis and after secretion. Here, we show using intrinsic and extrinsic fluorescence probes that Z α1AT exists in a non-native conformation. We examined the thermodynamic stability by transverse urea gradient gel electrophoresis, thermal denaturation and equilibrium guanidine hydrochloride unfolding and found that, despite structural differences between the two proteins, wild-type α1AT and Z α1AT display similar unfolding pathways and thermodynamic stabilities. Far-UV circular dichroism and bis-ANS (4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid, dipotassium salt) fluorescence suggest that the intermediate ensembles formed during unfolding of wild-type α1AT and Z α1AT are characterized by similar structural features. Kinetic analysis of the unfolding transition showed that Z α1AT unfolds at least 1.5-fold faster than the wild type. The biological implications of these data are discussed.