Cofactors are essential constituents of stable and seeding-active tau fibrils.

Amyloid fibrils are cross-ß-rich aggregates that are exceptionally stable forms of protein assembly. Accumulation of tau amyloid fibrils is involved in many neurodegenerative diseases, including Alzheimer's disease (AD). Heparin-induced aggregates have been widely used and assumed to be a good tau amyloid fibril model for most biophysical studies. Here we show that mature fibrils made of 4R tau variants, prepared with heparin or RNA, spontaneously depolymerize and release monomers when their cofactors are removed. We demonstrate that the cross-ß-sheet assembly formed in vitro with polyanion addition is unstable at room temperature. We furthermore demonstrate high seeding capacity with transgenic AD mouse brain-extracted tau fibrils in vitro that, however, is exhausted after one generation, while supplementation with RNA cofactors resulted in sustained seeding over multiple generations. We suggest that tau fibrils formed in brains are supported by unknown cofactors and inhere higher-quality packing, as reflected in a more distinct conformational arrangement in the mouse fibril-seeded, compared with heparin-induced, tau fibrils. Our study suggests that the role of cofactors in tauopathies is a worthy focus of future studies, as they may be viable targets for diagnosis and therapeutics.

Surface chemical heterogeneity modulates silica surface hydration.

An in-depth knowledge of the interaction of water with amorphous silica is critical to fundamental studies of interfacial hydration water, as well as to industrial processes such as catalysis, nanofabrication, and chromatography. Silica has a tunable surface comprising hydrophilic silanol groups and moderately hydrophobic siloxane groups that can be interchanged through thermal and chemical treatments. Despite extensive studies of silica surfaces, the influence of surface hydrophilicity and chemical topology on the molecular properties of interfacial water is not well understood. In this work, we controllably altered the surface silanol density, and measured surface water diffusivity using Overhauser dynamic nuclear polarization (ODNP) and complementary silica-silica interaction forces across water using a surface forces apparatus (SFA). The results show that increased silanol density generally leads to slower water diffusivity and stronger silica-silica repulsion at short aqueous separations (less than ∼4 nm). Both techniques show sharp changes in hydration properties at intermediate silanol densities (2.0-2.9 nm-2). Molecular dynamics simulations of model silica-water interfaces corroborate the increase in water diffusivity with silanol density, and furthermore show that even on a smooth and crystalline surface at a fixed silanol density, adjusting the spatial distribution of silanols results in a range of surface water diffusivities spanning ∼10%. We speculate that a critical silanol cluster size or connectivity parameter could explain the sharp transition in our results, and can modulate wettability, colloidal interactions, and surface reactions, and thus is a phenomenon worth further investigation on silica and chemically heterogeneous surfaces.

The Role of Backbone Polarity on Aggregation and Conduction of Ions in Polymer Electrolytes.

The usual understanding in polymer electrolyte design is that an increase in the polymer dielectric constant results in reduced ion aggregation and therefore increased ionic conductivity. We demonstrate here that in a class of polymers with extensive metal-ligand coordination and tunable dielectric properties, the extent of ionic aggregation is delinked from the ionic conductivity. The polymer systems considered here comprise ether, butadiene, and siloxane backbones with grafted imidazole side-chains, with dissolved Li+, Cu2+, or Zn2+ salts. The nature of ion aggregation is probed using a combination of X-ray scattering, electron paramagnetic resonance (in the case where the metal cation is Cu2+), and polymer field theory-based simulations. Polymers with less polar backbones (butadiene and siloxane) show stronger ion aggregation in X-ray scattering compared to those with the more polar ether backbone. The Tg-normalized ionic conductivities were however unaffected by the extent of aggregation. The results are explained on the basis of simulations which indicate that polymer backbone polarity does impact the microstructure and the extent of ion aggregation but does not impact percolation, leading to similar ionic conductivity regardless of the extent of ion aggregation. The results emphasize the ability to design for low polymer Tg through backbone modulation, separately from controlling ion-polymer interaction dynamics through ligand choice.

Water Dynamics from the Surface to the Interior of a Supramolecular Nanostructure.

Water within and surrounding the structure of a biological system adopts context-specific dynamics that mediate virtually all of the events involved in the inner workings of a cell. These events range from protein folding and molecular recognition to the formation of hierarchical structures. Water dynamics are mediated by the chemistry and geometry of interfaces where water and biomolecules meet. Here we investigate experimentally and computationally the translational dynamics of vicinal water molecules within the volume of a supramolecular peptide nanofiber measuring 6.7 nm in diameter. Using Overhauser dynamic nuclear polarization relaxometry, we show that drastic differences exist in water motion within a distance of about one nanometer from the surface, with rapid diffusion in the hydrophobic interior and immobilized water on the nanofiber surface. These results demonstrate that water associated with materials designed at the nanoscale is not simply a solvent, but rather an integral part of their structure and potential functions.

Overhauser dynamic nuclear polarization (ODNP)-enhanced two-dimensional proton NMR spectroscopy at low magnetic fields.

The majority of low-field Overhauser dynamic nuclear polarization (ODNP) experiments reported so far have been 1D NMR experiments to study molecular dynamics and in particular hydration dynamics. In this work, we demonstrate the application of ODNP-enhanced 2D J-resolved (JRES) spectroscopy to improve spectral resolution beyond the limit imposed by the line broadening introduced by the paramagnetic polarizing agent. Using this approach, we are able to separate the overlapping multiplets of ethyl crotonate into a second dimension and clearly identify each chemical site individually. Crucial to these experiments is interleaved spectral referencing, a method introduced to compensate for temperature-induced field drifts over the course of the NMR acquisition. This method does not require additional hardware such as a field-frequency lock, which is especially challenging when designing compact systems.

High-resolution Overhauser dynamic nuclear polarization enhanced proton NMR spectroscopy at low magnetic fields.

Dynamic nuclear polarization (DNP) has gained large interest due to its ability to increase signal intensities in nuclear magnetic resonance (NMR) experiments by several orders of magnitude. Currently, DNP is typically used to enhance high-field, solid-state NMR experiments. However, the method is also capable of dramatically increasing the observed signal intensities in solution-state NMR spectroscopy. In this work, we demonstrate the application of Overhauser dynamic nuclear polarization (ODNP) spectroscopy at an NMR frequency of 14.5 MHz (0.35 T) to observe DNP-enhanced high-resolution NMR spectra of small molecules in solutions. Using a compact hybrid magnet with integrated shim coils to improve the magnetic field homogeneity we are able to routinely obtain proton linewidths of less than 4 Hz and enhancement factors >30. The excellent field resolution allows us to perform chemical-shift resolved ODNP experiments on ethyl crotonate to observe proton J-coupling. Furthermore, recording high-resolution ODNP-enhanced NMR spectra of ethylene glycol allows us to characterize the microwave induced sample heating in-situ, by measuring the separation of the OH and CH2 proton peaks.

Tau-Cofactor Complexes as Building Blocks of Tau Fibrils.

The aggregation of the human tau protein into neurofibrillary tangles is directly diagnostic of many neurodegenerative conditions termed tauopathies. The species, factors and events that are responsible for the initiation and propagation of tau aggregation are not clearly established, even in a simplified and artificial in vitro system. This motivates the mechanistic study of in vitro aggregation of recombinant tau from soluble to fibrillar forms, for which polyanionic cofactors are the most commonly used external inducer. In this study, we performed biophysical characterizations to unravel the mechanisms by which cofactors induce fibrillization. We first reinforce the idea that cofactors are the limiting factor to generate ThT-active tau fibrils, and establish that they act as templating reactant that trigger tau conformational rearrangement. We show that heparin has superior potency for recruiting monomeric tau into aggregation-competent species compared to any constituent intermediate or aggregate "seeds." We show that tau and cofactors form intermediate complexes whose evolution toward ThT-active fibrils is tightly regulated by tau-cofactor interactions. Remarkably, it is possible to find mild cofactors that complex with tau without forming ThT-active species, except when an external catalyst (e.g., a seed) is provided to overcome the energy barrier. In a cellular context, we propose the idea that tau could associate with cofactors to form a metastable complex that remains "inert" and reversible, until encountering a relevant seed that can trigger an irreversible transition to ß-sheet containing species.

Conformation-based assay of tau protein aggregation.

Amyloid fiber-forming proteins are predominantly intrinsically disordered proteins (IDPs). The protein tau, present mostly in neurons, is no exception. There is a significant interest in the study of tau protein aggregation mechanisms, given the direct correlation between the deposit of ß-sheet structured neurofibrillary tangles made of tau and pathology in several neurodegenerative diseases, including Alzheimer's disease. Among the core unresolved questions is the nature of the initial step triggering aggregation, with increasing attention placed on the question whether a conformational change of the IDPs plays a key role in the early stages of aggregation. Specifically, there is growing evidence that a shift in the conformation ensemble of tau is involved in its aggregation pathway, and might even dictate structural and pathological properties of mature fibers. Yet, because IDPs lack a well-defined 3D structure and continuously exchange between different conformers, it has been technically challenging to characterize their structural changes on-pathway to aggregation. Here, we make a case that double spin labeling of the ß-sheet stacking region of tau combined with pulsed double electron-electron resonance spectroscopy is a powerful method to assay conformational changes occurring during the course of tau aggregation, by probing intramolecular distances around aggregation-prone domains. We specifically demonstrate the potential of this approach by presenting recent results on conformation rearrangement of the ß-sheet stacking segment VQIINK (known as PHF6*) of tau. We highlight a canonical shift of the conformation ensemble, on-pathway and occurring at the earliest stage of aggregation, toward an opening of PHF6*. We expect this method to be applicable to other critical segments of tau and other IDPs.

Active cancellation - A means to zero dead-time pulse EPR.

The necessary resonator employed in pulse electron paramagnetic resonance (EPR) rings after the excitation pulse and creates a finite detector dead-time that ultimately prevents the detection of signal from fast relaxing spin systems, hindering the application of pulse EPR to room temperature measurements of interesting chemical or biological systems. We employ a recently available high bandwidth arbitrary waveform generator (AWG) to produce a cancellation pulse that precisely destructively interferes with the resonant cavity ring-down. We find that we can faithfully detect EPR signal at all times immediately after, as well as during, the excitation pulse. This is a proof of concept study showcasing the capability of AWG pulses to precisely cancel out the resonator ring-down, and allow for the detection of EPR signal during the pulse itself, as well as the dead-time of the resonator. However, the applicability of this approach to conventional EPR experiments is not immediate, as it hinges on either (1) the availability of low-noise microwave sources and amplifiers to produce the necessary power for pulse EPR experiment or (2) the availability of very high conversion factor micro coil resonators that allow for pulse EPR experiments at modest microwave power.

DAC-board based X-band EPR spectrometer with arbitrary waveform control.

We present arbitrary control over a homogenous spin system, demonstrated on a simple, home-built, electron paramagnetic resonance (EPR) spectrometer operating at 8-10 GHz (X-band) and controlled by a 1 GHz arbitrary waveform generator (AWG) with 42 dB (i.e. 14-bit) of dynamic range. Such a spectrometer can be relatively easily built from a single DAC (digital to analog converter) board with a modest number of stock components and offers powerful capabilities for automated digital calibration and correction routines that allow it to generate shaped X-band pulses with precise amplitude and phase control. It can precisely tailor the excitation profiles "seen" by the spins in the microwave resonator, based on feedback calibration with experimental input. We demonstrate the capability to generate a variety of pulse shapes, including rectangular, triangular, Gaussian, sinc, and adiabatic rapid passage waveforms. We then show how one can precisely compensate for the distortion and broadening caused by transmission into the microwave cavity in order to optimize corrected waveforms that are distinctly different from the initial, uncorrected waveforms. Specifically, we exploit a narrow EPR signal whose width is finer than the features of any distortions in order to map out the response to a short pulse, which, in turn, yields the precise transfer function of the spectrometer system. This transfer function is found to be consistent for all pulse shapes in the linear response regime. In addition to allowing precise waveform shaping capabilities, the spectrometer presented here offers complete digital control and calibration of the spectrometer that allows one to phase cycle the pulse phase with 0.007° resolution and to specify the inter-pulse delays and pulse durations to ≤ 250 ps resolution. The implications and potential applications of these capabilities will be discussed.