Biophysical characterisation of the pathological Z variant of α1-Antitrypsin
thesisposted on 2017-02-17, 01:46 authored by Knaupp, Anja Sylvia
Protein misfolding is associated with a range of diseases and occurs when a protein meanders from its normal folding pathway resulting in the formation of a non-native state that can self-associate. One protein superfamily commonly associated with misfolding and disease are the serpins (serine proteinase inhibitors). Several members of this superfamily are prone to self-association which is linked to a range of diverse disorders including emphysema, liver disease, angioedema, neurodegeneration and thrombosis. The serpins are particularly susceptible to misfolding and aggregation as they are metastable which means their native state is not the thermodynamic minimum for the polypeptide chain. The inherent tension sustained in the native state is necessary for protease inhibition, however, mutations can easily result in conformational rearrangements which lead to polymeric states with significantly increased stability. α1-Antitrypsin (α1AT) is the serpin most commonly associated with misfolding and aggregation as there are several naturally occurring variants linked to disease. Z α1AT is the most common pathological variant of α1AT characterised by a glutamate to a lysine substitution at amino acid position 342 (Glu342Lys) which results in the loss of a salt bridge to Lys290. In healthy individuals, α1AT is expressed in hepatocytes and secreted into circulation to control the proteolytic activity of neutrophil elastase in the lower respiratory tract. The Z mutation leads to an increased propensity of α1AT to polymerise at its place of synthesis, the endoplasmic reticulum of hepatocytes, which is associated with loss-of-function and gain-of-toxic-function mechanisms. Why the Z mutation renders α1AT prone to polymerisation is not known and the data available on Z α1AT is limited and conflicting. This thesis has therefore focused on the in vitro characterisation of Z α1AT to determine the effect of the Z mutation on the α1AT molecule. An extensive biophysical analysis of Z α1AT was conducted and revealed that the molecule adopts an altered but functional conformation. In order to map the conformational change induced by the Z mutation onto the α1AT molecule single tryptophan mutants were created and subjected to an extensive fluorescence spectroscopic analysis. The single tryptophan data indicate that the Z mutation leads to a conformational rearrangement in the top of β-sheet A whereas the structural integrity of β-sheet B is unaffected. This structural rearrangement of β-sheet A does, however, not result in a significant destabilisation of Z α1AT in comparison to the wild-type. This observation is further supported by molecular dynamics simulations which suggest that the Z mutation results in the formation of new interactions that compensate for the lost interactions. Analysis of the role of the side chain charge of amino acid 342 revealed that it is not solely the loss of the 290-342 salt bridge but also the positive charge of Lys342 in Z α1AT that leads to the adoption of an alternative conformation and its increased propensity to polymerise. Together, the data presented in this thesis suggest that the Z mutation results in a decrease in the barrier height to aggregation-prone species and consequently in increased polymer formation.