Conformational change and intermediates in the unfolding of α1-antichymotrypsin

Serpins are the prototypical members of the conformational disease family, a group of proteins that undergoes a change in shape that subsequently leads to tissue deposition. One specific example is alpha(1)-antichymotrypsin (ACT), which undergoes misfolding and aggregation that has been implicated in emphysema and Alzheimer's disease. In this study we have used guanidine hydrochloride (GdnHCl)-induced denaturation to investigate the conformational changes involved in the folding and unfolding of ACT. When the reaction was followed by circular dichroism spectroscopy, one stable intermediate was observed in 1.5 M GdnHCl. The same experiment monitored by fluorescence revealed a second intermediate formed in 2.5 M GdnHCl. Both these intermediates bound the hydrophobic dye ANS. These data suggest a four-state model for ACT folding N <----> I-1 <----> I-2 <----> U. I-1 and I-2 both have a similar loss of secondary structure (20%) compared with the native state. In I-2 however, there is a significant loss of tertiary interactions as revealed by changes in fluorescence emission maximum and intensity. Kinetic analysis of the unfolding reaction indicated that the native state is unstable with a fast rate of unfolding in water of 0.4 s(-1). The implications of these data for both ACT function and associated diseases are discussed.