Probing the unfolding pathway of α1-antitrypsin

Abstract

Protein misfolding plays a role in the pathogenesis of many diseases. α1-Antitrypsin misfolding leads to the accumulation of long chain polymers within the hepatocyte, reducing its plasma concentration and predisposing the patient to emphysema and liver disease. In order to understand the misfolding process, it is necessary to examine the folding of α1-antitrypsin through the different structures involved in this process. In this study we have used a novel technique in which unique cysteine residues were introduced at various positions into α1-antitrypsin and fluorescently labeled with N,N'- dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)- ethylenediamine. The fluorescence properties of each protein were studied in the native state and as a function of guanidine hydrochloride-mediated unfolding. The studies found that α1-antitrypsin unfolded through a series of intermediate structures. From the position of the fluorescence probes, the fluorescence quenching data, and the molecular modeling, we show that unfolding of α1-antitrypsin occurs via disruption of the A and C β-sheets followed by the B β-sheet. The implications of these data on both α1- antitrypsin function and polymerization are discussed.