ELECTRON PARAMAGNETIC RESONANCE AS A TOOL TO DETERMINE PARAMAGNETIC METAL COORDINATION IN NATIVELY UNSTRUCTURED NEURONAL PROTEINS
Neurodegenerative diseases are defined as diseases wherein nerve cells are damaged or destroyed and are a major health concern. Millions around the world have Alzheimer's and Parkinson's diseases, causing untold suffering and costing billions of dollars annually. Understanding the pathogenesis of these diseases on a molecular level is crucial to developing cures.
Parkinson's disease destroys dopaminergic neurons in the substantia nigra pars compacta area of the brain, causing many movement related disorders and ultimately death. A hallmark of Parkinson's disease is the cytosolic, filimental inclusion known as the Lewy Body, which consists mostly of the protein α-synuclein. α-Synuclein is a 140-residue unstructured neural peptide that has been linked to the pathogenesis of Parkinson's disease through genetics and animal models. Genetic mutations arising in Parkinson's are rare, and the disease appears to be instigated more commonly by external influences such as pesticides and heavy metals, in particular copper and aluminum. In vitro studies have indirectly demonstrated interaction between α-synuclein and copper ions. However, previous attempts to characterize the monomeric, unstructured α-synuclein-Cu2+ interaction more directly have lead to some confusion and conflicting results regarding the stoichiometry, chelation structures and binding affinity.
Alzheimer's disease is characterized as a dementia and is the most common neurological disorder, primarily affecting the cognitive abilities leading to extreme confusion, aggressive behavior and difficulty speaking. The disease ultimately leads to death within seven years after diagnosis. A hallmark of Alzheimer's disease is extracellular deposits, termed senile plaques, consisting mostly of the aggregated 40-42 residue protein Aβ. These plaques are shown to have elevated levels of copper relative to the rest of the brain and Aβ binds copper strongly in its monomeric form. Indirect evidence has suggested that aggregated Aβ chelates copper with higher affinity than the monomeric form. Direct evidence of this phenomenon, though, is lacking.
In this dissertation we use electron paramagnetic resonance (EPR) to directly investigate how the proteins α-synuclein and aggregated Aβ interact with Cu2+ ions. EPR is a technique that is uniquely suited to probe the interaction of natively unstructured proteins with paramagnetic species such as Cu2+. EPR combined with site-directed mutagenesis is a powerful tool for directly determining the protein-paramagnetic species stoichiometry and protein residues involved in copper chelation. Dissociation constant based EPR competition studies directly measure the affinity of the species of interest for the paramagnetic ions. Using these techniques we have determined the stoichiometry, chelation structures and affinities for both unstructured (chapter 2) and membrane bound (chapter 3) α-synuclein. Furthermore, in chapter 4, EPR competition studies between aggregated Aβ and human serum albumin demonstrate that the aggregated form of Aβ is capable of outcompeting albumin for Cu2+ whereas monomeric Aβ is not. This information helps to shed light on both of these proteins disease pathogenesis and perhaps their natural function as well. This knowledge is critical for the development of new therapies for these diseases.