Anomalous Stiffness Changes of Tau Protein in X-ray Single Molecule Observations

Abstract

Alzheimer Disease (AD) is one of the most famous neurodegenerative disorders, and the neurofibrillary tangles serve as a hallmark of AD. They are composed of paired helical filaments of hyperphosphorylated Tau proteins, which are the microtubule-associated protein [1]. In order to understand the functions of a protein, we must determine its structures. But Tau protein's structure hasn’t been identified because it is famous for being one of the intrinsically disordered proteins (IDP), which lack stable tertiary structures or secondary structures [2].Here we observed Tau protein's fluctuations with the Diffracted X-ray tracking (DXT).This method is able to monitor the tilting and twisting motions of single protein molecules with nanometer resolution.In this method, gold nanocrystals are attached on target protein molecules to probe their intramolecular motions, and these proteins are irradiated with synchrotron X-ray to get time displacements of Laue diffraction patterns from a gold nanocrystal. As a synchrotron X-ray beamline, we used KEK NW-14A and SPring-8 BL40XU in Japan.In our experiment, in order to reveal the relation between an aggregation process and hyperphosphorylated Tau proteins, we phosphorylated wild-type and mutated recombinant Tau proteins with GSK-3β. Threonine and serine sites of these mutated Tau proteins were converted into Alanine in order not to be phosphorylated. And we found tau protein molecules were fluctuating between 0.3-1 nm in aqueous solution when a shutter speed was 36 ms/frame. More importantly, their fluctuations decreased after phosphorylated by GSK-3β. Finally, we specified the phosphorylation sites that affect structural fluctuations of Tau proteins.[1] A. Cavallini et al., J. Biol. Chem. 288, 23331-23347, (2013).[2] V. Uversky et al., Annu. Rev. Biophys. 37, 215-246, (2008).