Conformational Change and Intermediates in the Unfolding of α1-Antichymotrypsin

Abstract

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 ␣ 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 7 I 1 7 I 2 7 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.Conformational disorders, as defined by Carrell and colleagues (1, 2), arise when a protein undergoes a change in shape that leads to self-association, tissue deposition, and disease. The serpin superfamily includes some of the most studied proteins involved in this group of disorders. The misfolding and aggregation of serpins have been implicated in diseases ranging from emphysema and liver disease, to cancer and dementia (3-5). It is, however, extremely surprising that so little is known about the basic folding pathways of this class of protein.A number of studies have shown that ␣ 1 -antitrypsin unfolds through one intermediate (6 -8), and recent studies with maspin (9) and plasminogen activator inhibitor-1 (10) have shown similar results.The folding pathway of ␣ 1 -antichymotrypsin (ACT), 1 which is involved in regulation of several proteinases, including chymotrypsin, cathepsin G, and mast cell chymase, has, so far, received no attention. ACT is an acute phase DNA binding glycoprotein that possesses the characteristic serpin tertiary fold, composed of three ␤-sheets (A, B, and C) surrounded by nine ␣-helices (A-I) (11, 12). X-ray crystallographic and biochemical data have shown that ACT is capable of adopting several different conformations (12, 13). Cleavage of the reactive center loop (RCL) by noncognate proteinases causes full insertion of the RCL into the A ␤-sheet, forming an additional ␤-strand, which confers additional stability to the protein (12). The recent structure of an ACT variant demonstrated that partial insertion of the RCL was also possible, concomitant with unwinding and insertion of part of the F-helix, which lies on the A ␤-sheet (14). RCL sheet insertion is also possible in the absence o...

Preventing serpin aggregation: The molecular mechanism of citrate action upon antitrypsin unfolding

“…In these experiments, native AAT was unfolded in 5 M GdnHCl and the change in tryptophan fluorescence was monitored, over time, as a probe of conformational change. Consistent with previous studies (Pearce et al 2000), our data reveals that in the absence of citrate, a hyperfluorescent intermediate species is formed within the dead time of the machine, which then decays to the unfolded form with an apparent relaxation rate constant of 18.5 s À1 (Fig. 1).…”

Aeropin from the Extremophile Pyrobaculum aerophilum Bypasses the Serpin Misfolding Trap

“…While individually the fluorescence and CD unfolding curves suggest a two-state unfolding mechanism, the observation that they are not superimposable indicates that aeropin follows a three-state model with an unfolding intermediate. The presence of an unfolding intermediate has been previously characterized in several serpins by their ability to bind the hydrophobic dye, bis-ANS (33,42). To test whether the intermediate ensemble adopted by aeropin possessed such a property, we incubated denatured aeropin in the presence of bis-ANS, and recorded the resulting spectra.…”

The Selective Inhibition of Serpin Aggregation by the Molecular Chaperone, α-Crystallin, Indicates a Nucleation-dependent Specificity

“…It is feasible that the non-native species of ␣ 1 -AT and ACT may be sufficiently different in conformation to explain the specificity of ␣-crystallin interaction. However, given the high sequence homology and adoption of similar folding intermediates (29,35), it seems unlikely that the relatively subtle differences in structure between ACT and ␣ 1 -AT would have a major role in determining the substrate specificity of ␣-crystallin for ACT over ␣ 1 -AT particularly because of the broad substrate specificity of ␣-crystallin and other sHsps as determined from many studies of sHsp chaperone action (6 -8). Instead, a much more tangible contribution to specificity may be the distinct mechanistic and kinetic differences between the aggregation processes of the two serpins.…”

“…Molecular masses (MW) are in kDa. bis-ANS fluorescence when the polymerogenic precursor of ACT is formed (29). We examined the kinetics of this conformational change in the presence and absence of ␣-crystallin.…”