%0 Journal Article
%T Reactive centre loop mutants of ¦Á-1-antitrypsin reveal position-specific effects on intermediate formation along the polymerization pathway
%A Imran Haq
%A James£¿A. Irving
%A Sarah£¿V. Faull
%A Jennifer£¿A. Dickens
%J Bioscience Reports
%D 2013
%I Portland Press, Biochemical Society
%R 10.1042/bsr20130038
%X The common severe Z mutation (E342K) of ¦Á1-antitrypsin forms intracellular polymers that are associated with liver cirrhosis. The native fold of this protein is well-established and models have been proposed from crystallographic and biophysical data for the stable inter-molecular configuration that terminates the polymerization pathway. Despite these molecular ¡®snapshots¡¯, the details of the transition between monomer and polymer remain only partially understood. We surveyed the RCL (reactive centre loop) of ¦Á1-antitrypsin to identify sites important for progression, through intermediate states, to polymer. Mutations at P14P12 and P4, but not P10P8 or P2P1¡ä, resulted in a decrease in detectable polymer in a cell model that recapitulates the intracellular polymerization of the Z variant, consistent with polymerization from a near-native conformation. We have developed a FRET (F rster resonance energy transfer)-based assay to monitor polymerization in small sample volumes. An in vitro assessment revealed the position-specific effects on the unimolecular and multimolecular phases of polymerization: the P14P12 region self-inserts early during activation, while the interaction between P6P4 and ¦Â-sheet A presents a kinetic barrier late in the polymerization pathway. Correspondingly, mutations at P6P4, but not P14P12, yield an increase in the overall apparent activation energy of association from ~360 to 550 kJ mol 1.
%K cirrhosis
%K emphysema
%K FRET
%K intermediate
%K polymerization
%K serpin
%U http://www.bioscirep.org/bsr/033/e046/bsr033e046.htm