%0 Thesis %A Yang, Li %D 2017 %T Characterisation of a superstable consensus serpin, cAT %U https://bridges.monash.edu/articles/thesis/Characterisation_of_a_superstable_consensus_serpin_cAT/4669306 %R 10.4225/03/58ab827e4f4e2 %K Stable proteins %K Thermostable proteins %K α1-antitrypsin %K Protein engineering %K Protein aggregation %K Serpin %K ethesis-20160321-12544 %K Protein misfolding %K thesis(doctorate) %K Protein folding %K monash:168207 %K Emphysema %K Protease inhibitors %K Restricted access and full embargo %K Consensus design %K 1959.1/1252019 %K 2016 %X Serpins belong to a large family of protease inhibitors with one of the most conserved structures. They are part of a group of proteins that contradict the theory that the native fold is at the thermodynamic minimum. In fact, native serpins are marginally stable and can adopt alternative conformations to increase their stability. However, their activity is compromised in the process. By utilising the consensus sequence approach, through aligning 219 eukaryotic serpins and using the reactive centre loop of α1-antitrypsin, a consensus serpin (cAT) was designed. This synthetic serpin contains residues conserved in 70% of all the sequences used and is at the evolutionary centre of mass. The main objective of this thesis is to determine whether the consensus approach will yield a protein that behaves like a serpin through characterising the biochemical, biophysical and folding properties as well as investigating a disease mutation and a known surface cavity on the protein. cAT was firstly characterised via standard biochemical analyses. The protein exhibited many features observed in serpins. cAT exhibited activity that fell within the physiological range of a typical serpin. However, unlike most serpins, cAT displayed extremely high thermostability and remarkable resistance to polymerisation. In addition, the protein folded in an apparent two state folding mechanism via a folding intermediate. The creation of single tryptophan mutants allowed the intrinsic probe into the folding around the regions at helix F, H and the loop connecting β-sheets A and C. The resulting double tryptophan to phenylalanine mutations did not significantly alter the stability nor the activity of each mutant variant. Intrinsic fluorescent scans in high concentration of urea revealed that residual structure exists around helix H, therefore suggesting that forms part of the hydrophobic core as reported previously in other serpins. This further supports the structural and conformational similarity of cAT to other serpin proteins. The elucidation into the mechanism by which the Siiyama (S35F) mutation causes disease was possible in the hyper stable and polymer resistant cAT protein. As predicted, the Siiyama mutation caused a decrease in stability and substantially increased the rate of polymerisation. The X-ray crystal structure of the cleaved cATSiiyama exposed areas most vulnerable to the mutation and for the first time revealed the mechanism by which the mutation can cause the disease in serpin. Lastly, the study into the citrate binding site has exposed the conservation of the site on cAT. The site binds to citrate which resulted in an increase in stability and resistance to polymerisation in a linearly dependent manner. The mathematical function between monomer half-life and that of stability was also established. Moreover, these results revealed that the stability of cAT can further be increased. These findings suggest that the consensus method can be employed to produce a fully functional serpin protein with a substantially improved stability and increased resistance to polymerisation. Moreover, by using cAT as a model serpin, detrimental disease mutations and conserved surface cavities can be studied. %I Monash University