ABSTRACT
We present field-domain rapid-scan (RS) electron paramagnetic resonance (EPR) at 8.6T and 240GHz. To enable this technique, we upgraded a home-built EPR spectrometer with an FPGA-enabled digitizer and real-time processing software. The software leverages the Hilbert transform to recover the in-phase (I) and quadrature (Q) channels, and therefore the raw absorptive and dispersive signals, χ' and χ'', from their combined magnitude (I2+Q2). Averaging a magnitude is simpler than real-time coherent averaging and has the added benefit of permitting long-timescale signal averaging (up to at least 2.5×106 scans) because it eliminates the effects of source-receiver phase drift. Our rapid-scan (RS) EPR provides a signal-to-noise ratio that is approximately twice that of continuous wave (CW) EPR under the same experimental conditions, after scaling by the square root of acquisition time. We apply our RS EPR as an extension of the recently reported time-resolved Gd-Gd EPR (TiGGER) [Maity et al., 2023], which is able to monitor inter-residue distance changes during the photocycle of a photoresponsive protein through changes in the Gd-Gd dipolar couplings. RS, opposed to CW, returns field-swept spectra as a function of time with 10ms time resolution, and thus, adds a second dimension to the static field transients recorded by TiGGER. We were able to use RS TiGGER to track time-dependent and temperature-dependent kinetics of AsLOV2, a light-activated phototropin domain found in oats. The results presented here combine the benefits of RS EPR with the improved spectral resolution and sensitivity of Gd chelates at high magnetic fields. In the future, field-domain RS EPR at high magnetic fields may enable studies of other real-time kinetic processes with time resolutions that are otherwise difficult to access in the solution state.
ABSTRACT
The superior antifouling performance of zwitterionic materials is commonly linked to their hydration structure, in which tight surface binding of water molecules inhibits solute adsorption. However, there is comparatively little direct experimental data on the hydration water structure and dynamics around zwitterionic moieties, including the longer-range behavior of the hydration shell that modulates the approach of solutes to the polymer surface. This work experimentally probes the dynamics of the diffusing hydration water molecules around a series of zwitterion chemistries using Overhauser dynamic nuclear polarization relaxometry. Surprisingly, water dynamics measured within â¼1 nm of the zwitterions were minimally inhibited compared to those near uncharged hydrophilic or cationic side chains. Specific dissolved ions further enhance the water diffusivity near the zwitterions, rendering the hydration shell bulk water-like. These results that the hydration of a zwitterion surface is nearly indistinguishable from bulk water suggest that these surfaces are "invisible" to biological constituents in a manner tunable by the ionic environment and the chemical design of the zwitterionic surface.
ABSTRACT
Crystallization pathways are essential to various industrial, geological, and biological processes. In nonclassical nucleation theory, prenucleation clusters (PNCs) form, aggregate, and crystallize to produce higher order assemblies. Microscopy and X-ray techniques have limited utility for PNC analysis due to the small size (0.5-3 nm) and time stability constraints. We present a new approach for analyzing PNC formation based on 31P nuclear magnetic resonance (NMR) spin counting of vitrified molecular assemblies. The use of glassing agents ensures that vitrification generates amorphous aqueous samples and offers conditions for performing dynamic nuclear polarization (DNP)-amplified NMR spectroscopy. We demonstrate that molecular adenosine triphosphate along with crystalline, amorphous, and clustered calcium phosphate materials formed via a nonclassical growth pathway can be differentiated from one another by the number of dipolar coupled 31P spins. We also present an innovative approach for examining spin counting data, demonstrating that a knowledge-based fitting of integer multiples of cosine wave functions, instead of the traditional Fourier transform, provides a more physically meaningful retrieval of the existing frequencies. This is the first report of multiquantum spin counting of assemblies formed in solution as captured under vitrified DNP conditions, which can be useful for future analysis of PNCs and other aqueous molecular clusters.
ABSTRACT
Hydration water dynamics, structure, and thermodynamics are crucially important to understand and predict water-mediated properties at molecular interfaces. Yet experimentally and directly quantifying water behavior locally near interfaces at the sub-nanometer scale is challenging, especially at interfaces submerged in biological solutions. Overhauser dynamic nuclear polarization (ODNP) experiments measure equilibrium hydration water dynamics within 8-15 angstroms of a nitroxide spin probe on instantaneous timescales (10 picoseconds to nanoseconds), making ODNP a powerful tool for probing local water dynamics in the vicinity of the spin probe. As with other spectroscopic techniques, concurrent computational analysis is necessary to gain access to detailed molecular level information about the dynamic, structural, and thermodynamic properties of water from experimental ODNP data. We chose a model system that can systematically tune the dynamics of water, a water-glycerol mixture with compositions ranging from 0 to 0.3 mole fraction glycerol. We demonstrate the ability of molecular dynamics (MD) simulations to compute ODNP spectroscopic quantities, and show that translational, rotational, and hydrogen bonding dynamics of hydration water align strongly with spectroscopic ODNP parameters. Moreover, MD simulations show tight correlations between the dynamic properties of water that ODNP captures and the structural and thermodynamic behavior of water. Hence, experimental ODNP readouts of varying water dynamics suggest changes in local structural and thermodynamic hydration water properties.
ABSTRACT
The structure of coupled electron spin systems is of fundamental interest to many applications, including dynamic nuclear polarization (DNP), enhanced nuclear magnetic resonance (NMR), the generation of electron spin qubits for quantum information science (QIS), and quantitative studies of paramagnetic systems by electron paramagnetic resonance (EPR). However, the characterization of electron spin coupling networks is nontrivial, especially at high magnetic fields. This study focuses on a system containing high concentrations of trityl radicals that give rise to a DNP enhancement profile of 1H NMR characteristic of the presence of electron spin clusters. When this system is subject to selective microwave saturation through pump-probe ELectron DOuble Resonance (ELDOR) experiments, electron spin hyperpolarization is observed. We show that the generation of an out-of-equilibrium longitudinal dipolar order is responsible for the transient hyperpolarization of electron spins. Notably, the coupled electron spin system needs to form an AX-like system (where the difference in the Zeeman interactions of two spins is larger than their coupling interaction) such that selective microwave irradiation can generate signatures of electron spin hyperpolarization. We show that the extent of dipolar order, as manifested in the extent of electron spin hyperpolarization generated, can be altered by tuning the pump or probe pulse length, or the interpulse delay in ELDOR experiments that change the efficiency to generate or readout longitudinal dipolar order. Pump-probe ELDOR with selective saturation is an effective means for characterizing coupled electron spins forming AX-type spin systems that are foundational for DNP and quantum sensing.
ABSTRACT
Dynamic nuclear polarization (DNP) utilizing narrow-line electron spin clusters (ESCs) to achieve nuclear spin resonance matching (ESC-DNP) by microwave irradiation is a promising way to achieve NMR signal enhancements with a wide design scope requiring low microwave power at high magnetic field. Here we present the design for a trityl-based tetra-radical (TetraTrityl) to achieve DNP for 1H NMR at 7 T, supported by experimental data and quantum mechanical simulations. A slow-relaxing (T1e ≈ 1 ms) 4-ESC is found to require at least two electron spin pairs at <8 Å e-e spin distance to yield 1H ESC-DNP enhancement, while squeezing the rest of the e-e spin distances to <12 Å results in optimal 1H ESC-DNP enhancements. Fast-relaxing ESCs (T1e ≈ 10 µs) are found to require a weakly coupled narrow-line radical (sensitizer) to extract polarization from the ESC. These results provide design principles for achieving a power-efficient DNP at high field via ESC-DNP.
ABSTRACT
Cryoprotectants play a crucial role in preserving biological material, ensuring their viability during storage and facilitating crucial applications such as the conservation of medical compounds, tissues, and organs for transplantation. However, the precise mechanism by which cryoprotectants modulate the thermodynamic properties of water to impede the formation and growth of ice crystals, thus preventing long-term damage, remains elusive. This is evident in the use of empirically optimized recipes for mixtures that typically contain DMSO, glycerol, and various sugar constituents. Here, we use terahertz calorimetry, Overhauser nuclear polarization, and molecular dynamics simulations to show that DMSO exhibits a robust structuring effect on water around its methyl groups, reaching a maximum at a DMSO mole fraction of XDMSO = 0.33. In contrast, glycerol exerts a smaller water-structuring effect, even at higher concentrations (Scheme 1). These results potentially suggest that the wrapped water around DMSO's methyl group, which can be evicted upon ligand binding, may render DMSO a more surface-active cryoprotectant than glycerol, while glycerol may participate more as a viscogen that acts on the entire sample. These findings shed light on the molecular intricacies of cryoprotectant solvation behavior and have potentially significant implications for optimizing cryopreservation protocols.
ABSTRACT
Prion-like spread of disease-specific tau conformers is a hallmark of all tauopathies. A 19-residue probe peptide containing a P301L mutation and spanning the R2/R3 splice junction of tau folds and stacks into seeding-competent fibrils and induces aggregation of 4R, but not 3R tau. These tau peptide fibrils propagate aggregated intracellular tau over multiple generations, have a high ß-sheet content, a colocalized lipid signal, and adopt a well-defined U-shaped fold found in 4R tauopathy brain-derived fibrils. Fully atomistic replica exchange molecular dynamics (MD) simulations were used to compute the free energy landscapes of the conformational ensemble of the peptide monomers. These identified an aggregation-prohibiting ß-hairpin structure and an aggregation-competent U-fold unique to 4R tauopathy fibrils. Guided by MD simulations, we identified that the N-terminal-flanking residues to PHF6, which slightly vary between 4R and 3R isoforms, modulate seeding. Strikingly, when a single amino acid switch at position 305 replaced the serine of 4R tau with a lysine from the corresponding position in the first repeat of 3R tau, the seeding induced by the 19-residue peptide was markedly reduced. Conversely, a 4R tau mimic with three repeats, prepared by replacing those amino acids in the first repeat with those amino acids uniquely present in the second repeat, recovered aggregation when exposed to the 19-residue peptide. These peptide fibrils function as partial prions to recruit naive 4R tau-ten times the length of the peptide-and serve as a critical template for 4R tauopathy propagation. These results hint at opportunities for tau isoform-specific therapeutic interventions.
Subject(s)
Prions , Tauopathies , Humans , tau Proteins/metabolism , Tauopathies/metabolism , Protein Isoforms/metabolism , Prions/metabolism , Peptides , Amino AcidsABSTRACT
The separation and anti-fouling performance of water purification membranes is governed by both macroscopic and molecular-scale water properties near polymer surfaces. However, even for poly(ethylene oxide) (PEO) - ubiquitously used in membrane materials - there is little understanding of whether or how the molecular structure of water near PEO surfaces affects macroscopic water diffusion. Here, we probe both time-averaged bulk and local water dynamics in dilute and concentrated PEO solutions using a unique combination of experimental and simulation tools. Pulsed-Field Gradient NMR and Overhauser Dynamic Nuclear Polarization (ODNP) capture water dynamics across micrometer length scales in sub-seconds to sub-nanometers in tens of picoseconds, respectively. We find that classical models, such as the Stokes-Einstein and Mackie-Meares relations, cannot capture water diffusion across a wide range of PEO concentrations, but that free volume theory can. Our study shows that PEO concentration affects macroscopic water diffusion by enhancing the water structure and altering free volume. ODNP experiments reveal that water diffusivity near PEO is slower than in the bulk in dilute solutions, previously not recognized by macroscopic transport measurements, but the two populations converge above the polymer overlap concentration. Molecular dynamics simulations reveal that the reduction in water diffusivity occurs with enhanced tetrahedral structuring near PEO. Broadly, we find that PEO does not simply behave like a physical obstruction but directly modifies water's structural and dynamic properties. Thus, even in simple PEO solutions, molecular scale structuring and the impact of polymer interfaces is essential to capturing water diffusion, an observation with important implications for water transport through structurally complex membrane materials.
ABSTRACT
Amyloid aggregation of the intrinsically disordered protein (IDP) tau is involved in several diseases, called tauopathies. Some tauopathies can be inherited due to mutations in the gene encoding tau, which might favor the formation of tau amyloid fibrils. This work aims at deciphering the mechanisms through which the disease-associated single-point mutations promote amyloid formation. We combined biochemical and biophysical characterization, notably, small-angle X-ray scattering (SAXS), to study six different FTDP-17 derived mutations. We found that the mutations promote aggregation to different degrees and can modulate tau conformational ensembles, intermolecular interactions, and liquid-liquid phase separation propensity. In particular, we found a good correlation between the aggregation lag time of the mutants and their radii of gyration. We show that mutations disfavor intramolecular protein interactions, which in turn favor extended conformations and promote amyloid aggregation. This work proposes a new connection between the structural features of tau monomers and their propensity to aggregate, providing a novel assay to evaluate the aggregation propensity of IDPs.
ABSTRACT
Phosphate is an essential anion in the human body, comprising approximately 1% of the total body weight, and playing a vital role in metabolism, cell membranes, and bone formation. We have recently provided spectroscopic, microscopic, and computational evidence indicating that phosphates can aggregate much more readily in solution than previously thought. This prior work provided indirect evidence through the observation of unusual 31 P NMR relaxation and line-broadening effects with increasing temperature. Here, we show that, under conditions of slow exchange and selective RF saturation, additional features become visible in chemical exchange saturation transfer (CEST) experiments, which appear to be related to the previously reported phosphate clustering. In particular, CEST shows pronounced dips several ppm upfield of the main phosphate resonance at low temperatures, while direct 31 P spectroscopy does not produce any signals in that range. We study the pH dependence of these new spectroscopic features and present exchange and spectroscopic parameters based on fitting the CEST data. These findings could be of importance in the investigation of phosphate dynamics, especially in the biological milieu.
Subject(s)
Algorithms , Phosphates , Humans , Magnetic Resonance Imaging/methods , Magnetic Resonance Spectroscopy/methods , Hydrogen-Ion ConcentrationABSTRACT
Understanding the spatial distribution of the P1 centers is crucial for diamond-based sensors and quantum devices. P1 centers serve as polarization sources for dynamic nuclear polarization (DNP) quantum sensing and play a significant role in the relaxation of nitrogen vacancy (NV) centers. Additionally, the distribution of NV centers correlates with the distribution of P1 centers, as NV centers are formed through the conversion of P1 centers. We utilized DNP and pulsed electron paramagnetic resonance (EPR) techniques that revealed strong clustering of a significant population of P1 centers that exhibit exchange coupling and produce asymmetric line shapes. The 13C DNP frequency profile at a high magnetic field revealed a pattern that requires an asymmetric EPR line shape of the P1 clusters with electron-electron (e-e) coupling strengths exceeding the 13C nuclear Larmor frequency. EPR and DNP characterization at high magnetic fields was necessary to resolve energy contributions from different e-e couplings. We employed a two-frequency pump-probe pulsed electron double resonance technique to show cross-talk between the isolated and clustered P1 centers. This finding implies that the clustered P1 centers affect all of the P1 populations. Direct observation of clustered P1 centers and their asymmetric line shape offers a novel and crucial insight into understanding magnetic noise sources for quantum information applications of diamonds and for designing diamond-based polarizing agents with optimized DNP efficiency for 13C and other nuclear spins of analytes. We propose that room temperature 13C DNP at a high field, achievable through straightforward modifications to existing solution-state NMR systems, is a potent tool for evaluating and controlling diamond defects.
ABSTRACT
Intracerebral hemorrhage (ICH) is the second most common stroke subtype associated with high morbidity and mortality rates. Although various brain regions are susceptible to ICH, putaminal hemorrhage is the most common, whereas cortical ICH is less common. Here, we report the case of a 69-year-old man who developed a parietal cortical ICH. The patient developed hypoesthesia and paresthesia in the right upper lip and hand; however, the weakness was not severe. Twenty-five days after the ICH onset, the manual muscle test results were normal, but he had difficulty eating and shaving because of decreased hand dexterity. The rehabilitation focused on improving fine hand motor function and endurance. On the 94th day after ICH onset, paresthesia remained only in the fingertips, and the upper lip sensory change disappeared. Patients with sensory symptoms in the perioral area, hands, and brain lesions were previously referred to as having cheiro-oral syndrome (COS). With the advancement of neuroimaging, the use of this term has decreased, as cerebrovascular events can explain patient symptoms in correlation with neuroanatomy, etiology, and pathogenesis. We report a patient with cortical ICH, also known as COS, which is a stroke syndrome with a good prognosis.
ABSTRACT
Tau forms toxic fibrillar aggregates in a family of neurodegenerative diseases known as tauopathies. The faithful replication of tauopathy-specific fibril structures is a critical gap for developing diagnostic and therapeutic tools. This study debuts a strategy of identifying a critical segment of tau that forms a folding motif that is characteristic of a family of tauopathies and isolating it as a standalone peptide that form seeding-competent fibrils. The 19-residue jR2R3 peptide (295-313) spanning the R2/R3 splice junction of tau, in the presence of P301L, forms seeding-competent amyloid fibrils. This tau fragment contains the hydrophobic VQIVYK hexapeptide that is part of the core of every pathological tau fibril structure solved to-date and an intramolecular counter-strand that stabilizes the strand-loop-strand (SLS) motif observed in 4R tauopathy fibrils. This study shows that P301L exhibits a duality of effects: it lowers the barrier for the peptide to adopt aggregation-prone conformations and enhances the local structuring of water around the mutation site that facilitates site-specific dewetting and in-register stacking of tau to form cross ß-sheets. We solve a 3 Å cryo-EM structure of jR2R3-P301L fibrils with a pseudo 2 1 screw symmetry in which each half of the fibril's cross-section contains two jR2R3-P301L peptides. One chain adopts a SLS fold found in 4R tauopathies that is stabilized by a second chain wrapping around the SLS fold, reminiscent of the 3-fold and 4-fold structures observed in 4R tauopathies. These jR2R3-P301L fibrils are able to template full length tau in a prion-like fashion. Significance Statement: This study presents a first step towards designing a tauopathy specific aggregation pathway by engineering a minimal tau prion building block, jR2R3, that can template and propagate distinct disease folds. We present the discovery that P301L-among the widest used mutations in cell and animal models of Alzheimer's Disease-destabilizes an aggregation-prohibiting internal hairpin and enhances the local surface water structure that serves as an entropic hotspot to exert a hyper-localized effect in jR2R3. Our study suggests that P301L may be a more suitable mutation to include in modeling 4R tauopathies than for modelling Alzheimer's Disease, and that mutations are powerful tools for the purpose of designing of tau prion models as therapeutic tools.
ABSTRACT
Dynamic nuclear polarization (DNP) can amplify the solid-state nuclear magnetic resonance (NMR) signal by several orders of magnitude. The mechanism of DNP utilizing α,γ-bisdiphenylene-ß-phenylallyl (BDPA) variants as Polarizing Agents (PA) has been the subject of lively discussions on account of their remarkable DNP efficiency with low demand for microwave power. We propose that electron spin clustering of sulfonated BDPA is responsible for its DNP performance, as revealed by the temperature-dependent shape of the central DNP profile and strong electron-electron (e-e) crosstalk seen by Electron Double Resonance. We demonstrate that a multielectron spin cluster can be modeled with three coupled spins, where electron J (exchange) coupling between one of the e-e pairs matching the NMR Larmor frequency induces the experimentally observed absorptive central DNP profile, and the electron T1e modulated by temperature and magic-angle spinning alters the shape between an absorptive and dispersive feature. Understanding the microscopic origin is key to designing new PAs to harness the microwave-power-efficient DNP effect observed with BDPA variants.
ABSTRACT
Favorable relaxation processes, high-field spectral properties, and biological compatibility have made spin-7/2 Gd3+-based spin labels an increasingly popular choice for protein structure studies using high-field electron paramagnetic resonance. However, high-field relaxation and decoherence in ensembles of half-integer high-spin systems, such as Gd3+, remain poorly understood. We report spin-lattice (T1) and phase memory (TM) relaxation times at 8.6 T (240 GHz), and we present the first comprehensive model of high-field, high-spin decoherence accounting for both the electron spin concentration and temperature. The model includes four principal mechanisms driving decoherence: energy-conserving electron spin flip-flops, direct "T1" spin-lattice relaxation-driven electron spin flip processes, indirect T1-driven flips of nearby electron spins, and nuclear spin flip-flops. Mechanistic insight into decoherence can inform the design of experiments making use of Gd3+ as spin probes or relaxivity agents and can be used to measure local average interspin distances as long as 17 nm.
ABSTRACT
Prion-like spread of disease-specific tau conformers is a hallmark of all tauopathies. A 19-residue probe peptide containing a P301L mutation and spanning the R2/R3 splice junction of tau, folds and stacks into seeding-competent fibrils and induces aggregation of 4R, but not 3R tau. These tau peptide fibrils propagate aggregated intracellular tau over multiple generations, have a high ß-sheet content, a colocalized lipid signal, and adopt a well-defined U-shaped fold found in 4R tauopathy brain-derived fibrils. Fully atomistic replica exchange molecular dynamics (MD) simulations were used to compute the free energy landscapes of the conformational ensemble of the peptide monomers. These identified an aggregation-prohibiting ß-hairpin structure and an aggregation-competent U-fold unique to 4R tauopathy fibrils. Guided by MD simulations, we identified that the N-terminal-flanking residues to PHF6, which slightly vary between 4R and 3R isoforms, modulate seeding. Strikingly, when a single amino acid switch at position 305 replaced the serine of 4R tau with a lysine from the corresponding position in the first repeat of 3R tau, the seeding induced by the 19-residue peptide was markedly reduced. Conversely, a 4R tau mimic with three repeats, prepared by replacing those amino acids in the first repeat with those amino acids uniquely present in the second repeat, recovered aggregation when exposed to the 19-residue peptide. These peptide fibrils function as partial prions to recruit naïve 4R tau-ten times the length of the peptide-and serve as a critical template for 4R tauopathy propagation. These results hint at opportunities for tau isoform-specific therapeutic interventions.
ABSTRACT
Acorn (Quercus acutissima CARR.) has been used in traditional food and medicinal ethnopharmacology in Asia, and it has shown multifarious functions such as antidementia, antiobesity, and antiasthma functions. However, there is limited scientific evidence about the efficacy of acorn for ameliorating skin problems. Treatment with ethanol-extracted acorns (EeA's) ablated the expression of inducible nitric oxide synthase (iNOS), cyclooxygenase 2 (COX2), monocyte chemoattractant protein-1 (MCP-1), and interleukin (IL)-8 stimulated by tumor necrosis factor (TNF)-α in human adult low calcium high temperature (HaCaT) cells under sublethal dosages. In addition, treatment with EeA dose dependently inhibited the ex vivo hyper keratin formation induced by TNF-α in HaCaT cells in conjunction with the blockade of cytokeratin-1 (CK-1) and cytokeratin-5 (CK-5) expression. Moreover, EeA treatment stimulated the expression of hyaluronic acid (HA) expression in human fibroblasts in a dose-dependent manner. Linoleamide was identified as the functional component of EeA using preparative high-performance liquid chromatography and ultra high performance liquid chromatography-mass spectrometry-mass spectrometry analysis, and the anti-inflammatory features and enhanced HA expression were verified. Collectively, these results suggest the efficacy of EeA supplementation in improving skin problems via anti-inflammation and upregulating HA production.
Subject(s)
Hyaluronic Acid , Quercus , Adult , Humans , Keratinocytes , HaCaT Cells , EthanolABSTRACT
Glycerol is a major cryoprotective agent and is widely used to promote protein stabilization. By a combined experimental and theoretical study, we show that global thermodynamic mixing properties of glycerol and water are dictated by local solvation motifs. We identify three hydration water populations, i.e., bulk water, bound water (water hydrogen bonded to the hydrophilic groups of glycerol) and cavity wrap water (water hydrating the hydrophobic moieties). Here, we show that for glycerol experimental observables in the THz regime allow quantification of the abundance of bound water and its partial contribution to the mixing thermodynamics. Specifically, we uncover a 1 : 1 connection between the population of bound waters and the mixing enthalpy, which is further corroborated by the simulation results. Therefore, the changes in global thermodynamic quantity - mixing enthalpy - are rationalized at the molecular level in terms of changes in the local hydrophilic hydration population as a function of glycerol mole fraction in the full miscibility range. This offers opportunities to rationally design polyol water, as well as other aqueous mixtures to optimize technological applications by tuning mixing enthalpy and entropy based on spectroscopic screening.
ABSTRACT
The recent discovery by cryo-electron microscopy that the neuropatho-logical hallmarks of different tauopathies, including Alzheimer's disease, corticobasal degeneration (CBD), and progressive supranuclear palsy (PSP), are caused by unique misfolded conformations of the protein tau is among the most profound developments in neurodegenerative disease research. To capitalize on these discoveries for therapeutic development, one must achieve in vitro replication of tau fibrils that adopt the rep-resentative tauopathy disease folds - a grand challenge. To understand whether the commonly used, but imperfect, fragment of the tau pro-tein, K18, is capable of inducing specific protein folds, fibril seeds derived from CBD- and PSP-infected biosensor cells expressing K18, were used to achieve cell-free assembly of naïve, recombinant 4R tau into fibrils without the addition of any cofactors. Using Double Electron Electron Resonance (DEER) spectroscopy, we discovered that cell-passaged patho-logical seeds generate heterogeneous fibrils that are distinct between the CBD and PSP lysate-seeded fibrils, and are also unique from heparin-induced tau fibril populations. Moreover, the lysate-seeded fibrils contain a characteristic sub-population that resembles either the CBD or PSP disease fold, corresponding with the respective starting patient sam-ple. These findings indicate that CBD and PSP patient-derived fibrils retain strain properties after passaging through K18 reporter cells.