Alzheimer’s disease (AD) is a complex neurodegenerative disorder that manifests itself through neuronal death and loss of synaptic transmission. Its complex pathophysiology includes a double proteinopathy characterized by aggregation of the amyloid b-protein (Ab) and neurofibrillary tangles (NFT) of the microtubule-associated tau protein. Our group has effectively implemented high resolution mass spectrometry to study the interaction of amyloidogenic proteins with lysine specific molecular tweezer (MT) compound CLR01. CLR01 preferentially binds to Lys residues on unstructured proteins and modifies their assembly into non-toxic states. We have employed top-down MS methods to gain structural insight into tau and tau:CLR01 complexes. In addition, we have discovered that performing top-down ECD MS of noncovalent tau:CLR01 complex can reveal the site of inhibitor binding.<br/><br/>Preliminary ESI-MS data for intact tau proteins indicate that top-down MS will be amenable to the study of tau isoforms and their PTMs. Our top-down MS-based methods have been successfully used to characterize the effects of CLR01 binding to 4R tau protein (45.8 kDa) and tau fragment (11 kDa). ESI-MS spectra were obtained for the unmodified and phosphorylated 4R-repeat domain of tau fragment and tau/CLR01 complex in a 1:1 stoichiometric ratio. The intact protein-inhibitor complex was further subjected to ECD-MS to obtain sequence information, and pinpoint the sites of inhibitor binding. In agreement with NMR results, ECD-MS data point to CLR01 binding sites in the microtubule-binding region, spanning residues K293-K340, which includes a six-residue nucleating segment PHF6 (VQIVYK) implicated in aggregation. Since phosphorylation plays an important role in tau aggregation, we have also tested phosphorylated tau to map the sites of phosphorylation. ECD-MS confirmed phosphorylation at Ser-235.<br/><br/>Using ion mobility spectrometry, the Bower’s group at UCSB observed a decrease in higher order amyloid-β oligomers in the presence of CLR01. Our ion mobility experiments on the tau fragment revealed a shift towards a more compact structure in the presence of CLR01.

Using native top-down nESI FTICR-MS to characterize the interaction of tau protein with assembly modulator CLR01

2019 Spring National Meeting

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