Conformational transitions governing function and aggregation of enzymes
File(s)
Author(s)
Patel, Jayneil Raiesh
Type
Thesis
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.
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.
Version
Open Access
Date Issued
2016-09
Date Awarded
2017-03
Copyright Statement
Attribution NoDerivatives 4.0 International Licence (CC BY-ND)
Advisor
De Simone, Alfonso
Sponsor
Biotechnology and Biological Sciences Research Council (Great Britain)
Publisher Department
Life Sciences
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)