Abstract
Serpins are protease inhibitors whose most stable state is achieved upon transition of a central 5-stranded β-sheet to a 6-stranded form. Mutations, low pH, denaturants and elevated temperatures promote this transition, which can result in a growing polymer chain of inactive molecules. Different types of polymer are possible, but, experimentally only heat has been shown to generate polymers in vitro consistent with ex vivo pathological specimens. Many mutations that alter the rate of heat-induced polymerization have been described, but interpretation is problematic because discrimination is lacking between the effect of global changes in native stability and specific effects on structural mechanism. We show that the temperature midpoint (Tm) of thermal denaturation reflects the transition of α1-antitrypsin to the polymerization intermediate, and determine the relationship with fixed-temperature polymerization half-times (t0.5) in the presence of stabilizing additives [TMAO (trimethylamine N-oxide), sucrose and sodium sulfate], point mutations and disulfide bonds. Combined with a retrospective analysis of 31 mutants characterized in the literature, the results of the present study show that global changes to native state stability are the predominant basis for the effects of mutations and osmolytes on heat-induced polymerization, summarized by the equation: ln(t0.5,mutant/t0.5,wild-type)=0.34×ΔTm. It is deviations from this relationship that hold key information about the polymerization process.
Highlights
Serpins are globular proteins that predominantly act as protease inhibitors in a wide range of proteolytic cascades and environments
We show that the temperature midpoint (Tm) of thermal denaturation reflects the transition of α1antitrypsin to the polymerization intermediate, and determine the relationship with fixed-temperature polymerization halftimes (t0.5) in the presence of stabilizing additives [trimethylamine N-oxide (TMAO), sucrose and sodium sulfate], point mutations and disulfide bonds
We sought to characterize this relationship in detail, at several temperatures, in an attempt to distinguish the specific effect of mutations on structural mechanism from nonspecific effects on native state stability
Summary
Serpins are globular proteins that predominantly act as protease inhibitors in a wide range of proteolytic cascades and environments. It was found that increased thermal stability shared a strong direct correlation with a reduced rate of polymerization This relationship was confirmed by a range of engineered disulfides and two point mutants of α1-antitrypsin. The collective results demonstrate that changes in stability of the native state, and not specific interference with molecular mechanism, are the dominant cause of mutant-based effects on polymerization. In the case of gel images presented by Gilis et al [29], densitometry was performed retrospectively using GelAnalyzer 2010a software (http://gelanalyzer.com) One study used both gels and bis-ANS (4,4 -dianilino-1,1 -binaphthyl-5,5 -disulfonic acid) dye to monitor the polymerization reaction [50]. An examination of the rates they obtained reveals the closest linear correspondence between gel densitometry and the kcc value, the ‘rate of conformational change’ calculated from the rapid initial increase in bis-ANS fluorescence The kcc values presented by Cabrita et al [52] using bis-ANS, rather than the kagg values, were used as a basis for comparison (but were not included in the data used to derive the t0.5–Tm relationship)
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