Conformational transitions governing function and aggregation of enzymes

Abstract

In certain cases, the dynamical and conformational properties of enzymes, fundamental to their function, may lead to adverse effects such as loss of activity and aggregation. Studying these changes will help to further our understanding of how these structural transitions affect enzyme activity and solubility. Two exemplar enzymes are studied; Lipex is an industrially used lipase present in laundry detergent products, and HypF-N is used as a model system for protein aggregation. HypF-N can aggregate to form non-pathogenic amyloid fibrils, and under certain solution conditions can form toxic or non-toxic oligomers. NMR methods, suited to studying dynamical and conformational changes, were used. The aims of this thesis set to elucidate how solution conditions affect enzyme conformations. Using Lipex to understand how structural transitions affect functionality in gain or loss of activity. And through HypF-N understanding the process of aggregation and elucidating the origins of toxic gain of function. Lipex was expressed and purified in an Escherichia coli host. Activity assays demonstrated presence of surfactants and calcium can have positive and negative effects on catalytic activity. 2D HSQC NMR experiments showed differences in the conformation of Lipex between high-active and low-active conditions. NMR relaxation measurements indicated that a slower tumbling protein is linked to a more catalytically active enzyme. It was also determined that anionic surfactants cause destabilising effects to the tertiary structure of Lipex and should be reduced in laundry products to increase protein stability. NMR techniques demonstrated that aggregation occurs for HypF-N in folded and unfolded states. Despite these different initial conformations, a similar aggregation process exists across both toxic and non-toxic conditions. Solid-state NMR revealed a similar core structure in both toxic and non-toxic HypF-N oligomers. Differences in the mobility of the oligomer were detected using solid-state NMR and we hypothesise that certain flexible regions, and the interactions with the cell membrane, define toxicity.