Protein-misfolding diseases are based on a common principle of aggregation initiated by intra- and intermolecular contacts. The structural and conformational changes, induced by biochemical transformations, such as post-translational modifications (PTMs), often lead to protein unfolding and misfolding. Thus, these order-to-disorder or disorder-to-order transitions may regulate cellular function. Tau, a neuronal protein, regulates microtubule (MT) structure and overall cellular integrity. However, misfolded tau modified by PTMs results in MT destabilization, toxic tau aggregate formation, and ultimately cell death, leading to neurodegeneration. Currently, the lack of structural information surrounding tau severely limits understanding of neurodegeneration. This mini-review focuses on the current methodologies and approaches aimed at probing tau conformation and its role in various aspects of tau biochemistry. The recent applications of nuclear magnetic resonance, mass spectrometry, Förster resonance electron transfer, and molecular dynamics simulation toward structural analysis of conformational landscapes of tau will be described. The strategies developed for structural evaluation of tau may significantly improve our understanding of misfolding diseases.

The microtubule associated protein (MAP) Tau, primarily expressed in neurons, is known to have a variety of functions such as regulation of microtubule dynamics, modulation of kinesin motor motility and participation in signaling cascades. Research on the microtubule binding behavior of Tau reveals that Tau binds to the microtubule surface in an equilibrium between static and diffusive states. These functional states have been shown to be important in regulating kinesin motility during axonal transport. However, the structural relationship between the states has not been characterized. Therefore, using Total Internal Reflection Fluorescence (TIRF) microscopy, we developed a threecolor imaging assay to study the structural changes underlying Tau's dynamic behavior while bound to the microtubule surface using single molecule Fluorescence Resonance Energy Transfer (smFRET). Additionally, Alternating Laser Excitation (ALEX) is used to distinguish between single labeled populations and low FRET efficiency states. We have generated three 3RS-Tau FRET constructs to measure distances between distinct locations within Tau, between the N and C termini (N-C), between the microtubule binding repeats and the C terminal (3-C), and between the N terminal and the microtubule binding repeats (N-3). Initial studies indicate 3RS-Tau possesses distinct N and C termini interactions that allow for static versus diffusive binding. The examination of additional interactions will define overall structural changes in Tau on the microtubule surface. The smFRET-ALEX approach we have developed has applications for studying differences in other highly dynamic MAPs as well.

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