Virginia Meyer

Room-Temperature Distance Measurements of Immobilized Spin-Labeled Protein by DEER/PELDOR

Nitroxide spin labels are used for double electron-electron resonance (DEER) measurements of distances between sites in biomolecules. Rotation of gem-dimethyls in commonly used nitroxides causes spin echo dephasing times (Tm) to be too short to perform DEER measurements at temperatures between ∼80 and 295 K, even in immobilized samples. A spirocyclohexyl spin label has been prepared that has longer Tm between 80 and 295 K in immobilized samples than conventional labels. Two of the spirocyclohexyl labels were attached to sites on T4 lysozyme introduced by site-directed spin labeling. Interspin distances up to ∼4 nm were measured by DEER at temperatures up to 160 K in water/glycerol glasses. In a glassy trehalose matrix the Tm for the doubly labeled T4 lysozyme was long enough to measure an interspin distance of 3.2 nm at 295 K, which could not be measured for the same protein labeled with the conventional 1-oxyl-2,2,5,5-tetramethyl-3-pyrroline-3-(methyl)methanethio-sulfonate label.

Conformational Basis for Asymmetric Seeding Barrier in Filaments of Three- and Four-Repeat Tau

Tau pathology in Alzheimer’s disease is intimately linked to the deposition of proteinacious filaments, which akin to infectious prions, have been proposed to spread via seeded conversion. Here we use double electron–electron resonance (DEER) spectroscopy in combination with extensive computational analysis to show that filaments of three- (3R) and four-repeat (4R) tau are conformationally distinct. Distance measurements between spin labels in the third repeat, reveal tau amyloid filaments as ensembles of known β-strand–turn−β-strand U-turn motifs. Whereas filaments seeded with 3R tau are structurally homogeneous, filaments seeded with 4R tau are heterogeneous, composed of at least three distinct conformers. These findings establish a molecular basis for the seeding barrier between different tau isoforms and offer a new powerful approach for investigating the composition and dynamics of amyloid fibril ensembles.

Relaxation Times and Line Widths of Isotopically-Substituted Nitroxides in Aqueous Solution at X-band

Optimization of nitroxides as probes for EPR imaging requires detailed understanding of spectral properties. Spin lattice relaxation times, spin packet line widths, nuclear hyperfine splitting, and overall lineshapes were characterized for six low molecular weight nitroxides in dilute deoxygenated aqueous solution at X-band. The nitroxides included 6-member, unsaturated 5-member, or saturated 5-member rings, most of which were isotopically labeled. The spectra are near the fast tumbling limit with T(1)∼T(2) in the range of 0.50-1.1 μs at ambient temperature. Both spin-lattice relaxation T(1) and spin-spin relaxation T(2) are longer for (15)N- than for (14)N-nitroxides. The dominant contributions to T(1) are modulation of nitrogen hyperfine anisotropy and spin rotation. Dependence of T(1) on nitrogen nuclear spin state m(I) was observed for both (14)N and (15)N. Unresolved hydrogen/deuterium hyperfine couplings dominate overall line widths. Lineshapes were simulated by including all nuclear hyperfine couplings and spin packet line widths that agreed with values obtained by electron spin echo. Line widths and relaxation times are predicted to be about the same at 250 MHz as at X-band.

X-band rapid-scan EPR of samples with long electron spin relaxation times: a comparison of continuous wave, pulse and rapid-scan EPR

X-band room temperature spectra obtained by rapid-scan, continuous wave, field-swept echo-detected and Fourier transform electron paramagnetic resonance (FTEPR) were compared for three samples with long electron spin relaxation times: amorphous hydrogenated silicon (T1 = 11 μs, T2 = 3.3 μs), 0.2% N@C60 solid (T1 = 120–160 μs, T2 = 2.8 μs) and neutral single substitutional nitrogen centres (NS0) in diamonds (T1 = 2300 μs, T2 = 230 μs). For each technique, experimental parameters were selected to give less than 2% broadening of the lineshape. For the same data acquisition times, the signal-to-noise for the rapid-scan spectra was one-to-two orders of magnitude better than for continuous wave or field-swept echo-detected spectra. For amorphous hydrogenated silicon, T2* (∼ 10 ns) is too short to perform FTEPR. For 0.2% N@C60, the signal-to-noise ratio for rapid scan is about five times better than for FTEPR. For NS0 the signal-to-noise ratio is similar for rapid scan and FTEPR.

Amplification of Tau fibrils from minute quantities of seeds.

The propagation of Tau pathology in Alzheimer’s disease (AD) is thought to proceed through templated conversion of Tau protein into fibrils and cell-to-cell transfer of elongation-competent seeds. To investigate the efficiency of Tau conversion, we adapted the protein misfolding cyclic amplification assay used for the conversion of prions. Utilizing heparin as a cofactor and employing repetitive cycles of shearing and growth, synthetic Tau fibrils and Tau fibrils in AD brain extract are progressively amplified. Concurrently, self-nucleation is suppressed. The results highlight breakage-induced replication of Tau fibrils as a potential facilitator of disease spread.

Single Mutations in Tau Modulate the Populations of Fibril Conformers through Seed Selection

Seeded conversion of tau monomers into fibrils is a central step in the progression of tau pathology in Alzheimers disease and other neurodegenerative disorders. Self-assembly is mediated by the microtubule binding repeats in tau. There are either three or four repeats present depending on the protein isoform. Here, double electron-electron resonance spectroscopy was used to investigate the conformational ensemble of four-repeat tau fibrils. Single point mutations at key positions in the protein (ΔK280, P301S, P312I, D314I) markedly change the distribution of fibril conformers after template-assisted growth, whereas other mutations in the protein (I308M, S320F, G323I, G326I, Q336R) do not. These findings provide unprecedented insights into the seed selection of tau disease mutants and establish conformational compatibility as an important driving force in tau fibril propagation.

Electron spin-lattice relaxation mechanisms of rapidly-tumbling nitroxide radicals.

Electron spin relaxation times at 295 K were measured at frequencies between 250 MHz and 34 GHz for perdeuterated 2,2,6,6-tetramethyl-4-piperidone-1-oxyl (PDT) in five solvents with viscosities that result in tumbling correlation times, τR, between 4 and 50 ps and for three (14)N/(15)N pairs of nitroxides in water with τR between 9 and 19 ps. To test the impact of structure on relaxation three additional nitroxides with τR between 10 and 26 ps were studied. In this fast tumbling regime T2(-1)~T1(-1) at frequencies up to about 9 GHz. At 34 GHz T2(-1)>T1(-1) due to increased contributions to T2(-1) from incomplete motional averaging of g-anisotropy, and T2(-1)-T1(-1) is proportional to τR. The contribution to T1(-1) from spin rotation is independent of frequency and decreases as τR increases. Spin rotation dominates T1(-1) at 34 GHz for all τR studied, and at all frequencies studied for τR=4 ps. The contribution to T1(-1) from modulation of nitrogen hyperfine anisotropy increases as frequency decreases and as τR increases; it dominates at low frequencies for τR>~15 ps. The contribution from modulation of g anisotropy is significant only at 34 GHz. Inclusion of a thermally-activated process was required to account for the observation that for most of the radicals, T1(-1) was smaller at 250 MHz than at 1-2 GHz. The significant (15)N/(14)N isotope effect, the small H/D isotope effect, and the viscosity dependence of the magnitude of the contribution from the thermally-activated process suggest that it arises from intramolecular motions of the nitroxide ring that modulate the isotropic A values.

Electron spin relaxation and heterogeneity of the 1:1 α,γ-bisdiphenylene-β-phenylallyl (BDPA)/benzene complex.

The electron spin-spin relaxation time (T(2)) for the 1:1 crystalline complex of α,γ-bisdiphenylene-β-phenylallyl (BDPA) with benzene was determined by continuous wave (CW) and rapid scan electron paramagnetic resonance (EPR). T(2) for individual BDPA particles found by simulation of rapid scan spectra or by simulation of the Lorentzian line shapes of CW spectra were in good agreement. The T(2) for small BDPA particles in air ranged from 80 to 160 ns, which corresponds to peak-to-peak Lorentzian linewidths of 0.82-0.41 G. The removal of oxygen from the samples had a greater impact on the line width for particles that had shorter T(2) in air. Heterogeneity in the g-value was not observed at X-band. Scanning electron microscope (SEM) images showed that the BDPA particles had varying morphology.

Frequency dependence of electron spin relaxation times in aqueous solution for a nitronyl nitroxide radical and perdeuterated-tempone between 250 MHz and 34 GHz

Electron spin relaxation times of perdeuterated tempone (PDT) 1 and of a nitronyl nitroxide (2-(4-carboxy-phenyl)-4,4,5,5-tetramethylimidazoline-3-oxide-1-oxyl) 2 in aqueous solution at room temperature were measured by 2-pulse electron spin echo (T(2)) or 3-pulse inversion recovery (T(1)) in the frequency range of 250 MHz to 34 GHz. At 9 GHz values of T(1) measured by long-pulse saturation recovery were in good agreement with values determined by inversion recovery. Below 9 GHz for 1 and below 1.5 GHz for 2,T(1)~T(2), as expected in the fast tumbling regime. At higher frequencies T(2) was shorter than T(1) due to incomplete motional averaging of g and A anisotropy. The frequency dependence of 1/T(1) is modeled as the sum of spin rotation, modulation of g and A-anisotropy, and a thermally-activated process that has maximum contribution at about 1.5 GHz. The spin lattice relaxation times for the nitronyl nitroxide were longer than for PDT by a factor of about 2 at 34 GHz, decreasing to about a factor of 1.5 at 250 MHz. The rotational correlation times, τ(R) are calculated to be 9 ps for 1 and about 25 ps for 2. The longer spin lattice relaxation times for 2 than for 1 at 9 and 34 GHz are due predominantly to smaller contributions from spin rotation that arise from slower tumbling. The smaller nitrogen hyperfine couplings for the nitronyl 2 than for 1 decrease the contribution to relaxation due to modulation of A anisotropy. However, at lower frequencies the slower tumbling of 2 results in a larger value of ωτ(R) (ω is the resonance frequency) and larger values of the spectral density function, which enhances the contribution from modulation of anisotropic interactions for 2 to a greater extent than for 1.

Fracture and Growth Are Competing Forces Determining the Fate of Conformers in Tau Fibril Populations

Tau fibrils are pathological aggregates that can transfer between neurons and then recruit soluble Tau monomers by template-assisted conversion. The propagation of different fibril polymorphs is thought to be a contributing factor to phenotypic diversity in Alzheimer disease and other Tauopathies. We found that a homogeneous population of Tau fibrils composed of the truncated version K18 (residues 244–372) gradually converted to a new set of fibril conformers when subjected to multiple cycles of seeding and growth. Using double electron-electron resonance (DEER) spectroscopy, we observed that the distances between spin labels at positions 311 and 328 in the fibril core progressively decreased. The findings were corroborated by changes in turbidity, morphology, and protease sensitivity. Fibrils that were initially formed under stirring conditions exhibited an increased fragility compared with fibrils formed quiescently after multiple cycles of seeding. The quiescently formed fibrils were marked by accelerated growth. The difference in fragility and growth between the different conformers explains how the change in incubation condition could lead to the amplification of a minor subpopulation of fibrils. Under quiescent conditions where fibril breakage is minimal, faster growing fibrils have a selective advantage. The findings are of general importance as they suggest that changes in selective pressures during fibril propagation in the human brain could result in the emergence of new fibril conformers with varied clinicopathological consequences.