In vitro 0N4R tau fibrils contain a monomorphic β-sheet core enclosed by dynamically heterogeneous fuzzy coat segments

Abstract

Misfolding of the microtubule-binding protein tau into filamentous aggregates is characteristic of many neurodegenerative diseases such as Alzheimer’s disease and progressive supranuclear palsy. Determining the structures and dynamics of these tau fibrils is important for designing inhibitors against tau aggregation. Tau fibrils obtained from patient brains have been found by cryo-electron microscopy to adopt disease-specific molecular conformations. However, in vitro heparin-fibrillized 2N4R tau, which contains all four microtubule-binding repeats (4R), was recently found to adopt polymorphic structures. Here we use solid-state NMR spectroscopy to investigate the global fold and dynamics of heparin-fibrillized 0N4R tau. A single set of 13C and 15N chemical shifts was observed for residues in the four repeats, indicating a single β-sheet conformation for the fibril core. This rigid core spans the R2 and R3 repeats and adopts a hairpin-like fold that has similarities to but also clear differences from any of the polymorphic 2N4R folds. Obtaining a homogeneous fibril sample required careful purification of the protein and removal of any proteolytic fragments. A variety of experiments and polarization transfer from water and mobile side chains indicate that 0N4R tau fibrils exhibit heterogeneous dynamics: Outside the rigid R2–R3 core, the R1 and R4 repeats are semirigid even though they exhibit β-strand character and the proline-rich domains undergo large-amplitude anisotropic motions, whereas the two termini are nearly isotropically flexible. These results have significant implications for the structure and dynamics of 4R tau fibrils in vivo.

Alzheimer’s disease-relevant tau modifications selectively impact neurodegeneration and mitophagy in a novelC. eleganssingle-copy transgenic model

“…Here, in an attempt to circumvent potential caveats related to overexpression, novel transgenic AD models were engineered using single-copy Mos-transposon mediated insertion of a tau expression cassette into the worm genome (36, 42). The mec-7 promoter was used to drive the expression of 0N4R tau (59, 60) as a translational fusion with the fluorescent protein Dendra2 (40) in mechanosensory touch neurons ALML/ALMR, AVM, PLML/PLMR, and PVM (Fig. 1E-H), which mediate the behavioral response to light touch.…”

Pulsed Third-Spin-Assisted Recoupling NMR for Obtaining Long-Range13C-13C and15N-13C Distance Restraints

“…5e, left), but the transfer efficiency is much more sensitive to the 13 C rf field strength: significantly higher transfer efficiency is found at a 13 C rf field of 50 kHz than at 49 kHz, while 1 H and 15 N rf fields are held constant at 59.5 kHz and 50 kHz, respectively. The same modest 13 C rf field change also changed the offset at which maximum transfer occurs, from 60 ppm to 30 ppm. Thus, PAIN CP is extremely sensitive to both the rf field strength and the chemical shift offset.…”

“…For 2D 13 C- 13 C PULSAR experiments, the direct acquisition times were 21.5 ms and 16.1 ms for the 600 and 800 MHz spectra, respectively, whereas the indirect dimension 13 C evolution times were 9.9 ms and 7.4 ms, respectively. For the 2D 15 N-13 C PERSPIRATION CP experiments, the 13 C acquisition time was 11.9 ms and the 15 N evolution time was 10.2 ms. A pseudo-3D PERSPIRATION CP experiment was conducted on the 800 MHz spectrometer in which one indirect dimension varied the 13 C rf carrier from 0-200 ppm in 2.5 ppm increments, and the other indirect dimension varied the spin-lock pulse flip angles for both 13 C and 15 N from 0 to 360º in 15º increments. For the spin-lock pulses, the 13 C and 15 N rf field strengths were 50 kHz and 38.8 kHz, respectively, while the 1 H CW rf irradiation was 56-59 kHz during PULSAR and PERSPIRATION CP polarization transfer.…”

“…For clarity, we first analyze PERSPIRATION CP; explanations on how the analysis changes for PULSAR are given in the main text and Supporting Information. We consider a three-spin system consisting of one 1 H, one 15 N and one 13 C. The 1 H spin is subjected to CW rf irradiation while d-function pulses with flip angles qC and qN are applied at the center of each rotor period to 13 C and 15 N, respectively (Fig. 1a,c).…”

“…Magic-angle-spinning (MAS) solid-state nuclear magnetic resonance (SSNMR) spectroscopy produces high-resolution spectra by averaging orientation-dependent nuclear spin interactions such as chemical shift anisotropy (CSA), dipolar couplings, and quadrupolar couplings. MAS SSNMR is uniquely suited among spectroscopic techniques to provide atomic-resolution structural information of insoluble, non-crystalline biological macromolecules such as membrane proteins [1][2][3][4] , amyloid fibrils [5][6][7][8][9][10][11][12][13] , and cell walls [14][15][16][17] . Once chemical shift assignment is complete, three-dimensional structure determination predominantly relies on inter-atomic distance restraints.…”