title: Altered native stability is the dominant basis for susceptibility of α1-antitrypsin mutants to polymerization.
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.
authors.affiliation: *Cambridge Institute for Medical Research, Department of Medicine, University of Cambridge, Wellcome Trust/MRC Building, Hills Road, Cambridge CB2 0XY, U.K.
authors.forename: James A
authors.initials: JA
authors.lastname: Irving
authors.affiliation: †Wolfson Institute for Biomedical Research, The Cruciform Building, University College London, Gower Street, London WC1E 6BT, U.K.
authors.forename: Imran
authors.initials: I
authors.lastname: Haq
authors.affiliation: *Cambridge Institute for Medical Research, Department of Medicine, University of Cambridge, Wellcome Trust/MRC Building, Hills Road, Cambridge CB2 0XY, U.K.
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Dickens
authors.affiliation: *Cambridge Institute for Medical Research, Department of Medicine, University of Cambridge, Wellcome Trust/MRC Building, Hills Road, Cambridge CB2 0XY, U.K.
authors.forename: Sarah V
authors.initials: SV
authors.lastname: Faull
authors.affiliation: †Wolfson Institute for Biomedical Research, The Cruciform Building, University College London, Gower Street, London WC1E 6BT, U.K.
authors.forename: David A
authors.initials: DA
authors.lastname: Lomas
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D002417:Cattle; D002918:Chymotrypsin / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D002942:Circular Dichroism; D017354:Point Mutation / Q000235:genetics*; D058105:Polymerization; D055550:Protein Stability; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012189:Retrospective Studies; D013696:Temperature; D000515:alpha 1-Antitrypsin / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D000515:alpha 1-Antitrypsin; D002918:Chymotrypsin; C117608:alpha-chymotrypsin