Synopsis The common severe Z mutation (E342K) of α 1 -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

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... (reactive centre loop) of α 1 -antitrypsin to identify sites important for progression, through intermediate states, to polymer. Mutations at P 14 P 12 and P 4 , but not P 10 P 8 or P 2 P 1 , 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 (Förster 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 P 14 P 12 region self-inserts early during activation, while the interaction between P 6 P 4 and β-sheet A presents a kinetic barrier late in the polymerization pathway. Correspondingly, mutations at P 6 P 4 , but not P 14 P 12 , yield an increase in the overall apparent activation energy of association from ∼360 to 550 kJ mol − 1 . Reactive centre loop mutants of α-1-antitrypsin reveal position-specific effects on intermediate formation along the polymerization pathway. Biosci. Rep. 33(3), art:e00046.