Project Summary/Abstract Neurodegenerative diseases such as Alzheimer's disease are characterized by the abnormal deposition of fibrillar aggregates in the brain. The microtubule-associated protein tau can form such assemblies and defines a diverse group of diseases termed tauopathies. The prevalence of tau fibrils strongly correlates with disease progression, with mutations that increase aggregation propensity directly linked to disease. Even so, tau is thermostable and does not form fibrils in vitro or in cells without specific inducers. The intrinsically disordered nature of tau protein allows the sampling of multivalent interactions, facilitating the biological activity of microtubule binding. However, there is a gap in knowledge of how tau structurally transitions from aggregation- resistant to an aggregation-prone conformation. This proposal aims to fill this gap in knowledge by studying local interactions involving amyloid motifs in the aggregation-prone repeat domain of tau. I will leverage the differences in fibril toxicity of two isoforms of tau to create a map of sequence properties that define the fibrillization properties of tau. I hypothesize that interactions between amyloid motifs and their surrounding sequence mediate tau aggregation propensity; therefore, stabilizing these interactions to form local structures can prevent fibril formation. In this proposal, I will identify atomic interactions in tau that modulate aggregation propensity using molecular dynamics simulations combined with peptide aggregation assay. Then I will engineer modified tau constructs that are predicted to stabilize the local structure. Lastly, I will determine the local structure of the stabilized tau species using cross-linking mass spectrometry. Then I will test whether the stabilized structure maintains flexibility in the microtubule-binding motifs through in vitro and in cell microtubule-binding assays. This integrative approach will create a map of stabilizing local interactions within a tau monomer to design aggregation-resistant conformations of tau that retain biological microtubule-binding activity. By understanding the biophysical basis behind early misfolding events in tau, this project can inform the future design of diagnostics and therapeutics that stabilize non-toxic tau species to treat the diverse family of tauopathies.