Folding In Pieces

Circulating inhibitory serpins regulate important physiological processes including inflammation. Misfolding and polymerization of serpins in the endoplasmic reticulum (ER) can lead to both loss-of-function and gain-of-function diseases. The topology of the two-domain, non-sequential serpin fold is complicated with both the αβ and mainly β domains containing regions from near the N- and the C-termini. Despite these complications recent molecular dynamics simulations of serpin folding suggest that significant amounts of local secondary structure forms and rearranges for long-range contact formation resulting in the folded, active serpin. This folding pathway requires a balance between local structural stability and the flexibility/dynamics needed for long-range contact formation. To help test this proposed mechanism, we expressed and characterized N- to C-terminal fragments of the canonical, 394 residue serpin α1-antitrypsin (Alpha1). Even the shortest, 150 residue long, fragment showed cooperative unfolding and surprising stability with a denaturation midpoint of 1.9 M guanidinium hydrochloride. Nonetheless, the far-UV circular dichroism (CD) spectra differ from the spectra predicted for the full-length, native protein. Similarly, fluorescence emission of the longest fragment, 1-323, exhibited the hyper-fluorescent signature of the unfolding/folding intermediate observed during equilibrium denaturation or refolding of full-length Alpha1. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) was used to better define the structure of the fragments. After 10 seconds of deuteration, significant protection is observed in parts of the N-terminal region of the αβ domain, and for longer fragments in some of the β strands that form the core of the β domain. These results suggest that surprisingly stable local structures can be formed by fragments of the serpin Alpha1 and are consistent with models for serpin folding derived from molecular dynamics simulations. The results also suggest that significant amounts of serpin structure might form co-translationally.