Probing the interactions of intrinsically disordered proteins with copper ions and lipid membranes by fluorescence
Creators: name: Teng, Xiangyu
Publisher: name: Imperial College London
Year: 2021

description: Amyloid-β (Aβ) and α-synuclein (αSyn) are intrinsically disordered proteins (IDPs) with limited knowledge of normal functions. Their properties of interacting with metal ions and lipid membranes could possess physiological significance, but they may also induce pathological effects such as aggregation and reactive oxygen species (ROS) generation, thereby leading to the development of Alzheimer’s disease (AD) and Parkinson’s disease (PD). This thesis aims to unveil the molecular mechanisms of the interactions of Aβ and αSyn with Cu2+ and neuronal membranes, thus providing new information about the physiological and pathological roles of Aβ and αSyn, as well as Cu2+ and synaptic vesicle in neuronal synapses. An introduction of Aβ and αSyn, as well as their interactions with Cu2+ and lipid membranes is described in Chapter 1. Subsequently, in Chapter 2, the methods and materials used in this work are introduced. The main results of this thesis are presented from Chapter 3, in which the kinetic interactions of Cu2+ with phosphorylated serine residue 8 Aβ (pS8-Aβ) and N-truncated Aβ4-16 are presented. In this Chapter, the rate constants of Cu2+ binding to pS8-Aβ and N-truncated Aβ4-16 were both determined to be ~108 M-1 s-1 , which are comparable to that of wild type (WT) Aβ16. However, the binding affinity of Cu2+ to Aβ4-16 is much tighter (~103 - 104 times) than those of pS8-Aβ and WT-Aβ16. An interaction mechanism was proposed to describe the hierarchical binding of Cu2+ to Aβ4-16. The thesis then moves onto the study of αSyn. In Chapter 4, the production protocol of various fluorescently labelled and unlabelled αSyn samples is described first. The produced αSyn samples were then used in the work of Chapter 5 and Chapter 6 for investigating αSyn-Cu2+ interactions and αSyn-lipid interactions, respectively. In Chapter 5, the binding affinities and modes of Cu2+ to WT-αSyn and N-terminally acetylated (NAc) αSyn are presented. It was found that WT-αSyn shows around four orders of magnitude stronger binding affinity to Cu2+ than NAc-αSyn. Nevertheless, as described in Chapter 6, WT-αSyn shows weaker binding affinity with 50 nm synapticlike small unilamellar vesicle (SUV) than NAc-αSyn. In addition, the binding conformation of WT-αSyn on SUV was found to be mixed extended, curved or antiparallel helical structures when single αSyn binds to one SUV. However, if there are multiple αSyn molecules on one SUV, they tend to adopt the curved helical structure for association. In Chapter 7, SUV is shown to be able to bind Cu2+ as well, with a high affinity (Kd = 0.7(1) nM) which is even stronger than those of Aβ and αSyn. Such strong interaction is due to the constituent DOPS. In addition, the SUV-Cu(II) complex was found to be reduction-inert for ascorbate, but reactive for glutathione. Finally, in Chapter 8, the interactions among αSyn, Cu2+ and lipid membranes were investigated. Based on these results, a systematic hypothesis describing how αSyn, Cu2+ and synaptic vesicle collaboratively work in neurotransmission has been proposed. Overall, this thesis investigates how specified IDPs (Aβ and αSyn), Cu2+ and lipid membranes interact with each other from both kinetic and structural points of view. The results lead to a new hypothesis about the physiological functions of these elements, which may provide new reference points for underpinning the inner working of such an intertwined interacting network which potentially plays a significant role in neurotransmission and governs copper homeostasis in the synapse.