Characterization of Alzheimer's-like paired helical filaments from the core domain of tau protein using solid-state NMR spectroscopy

The polymerization of the microtubule-associated protein tau into paired helical filaments (PHFs) represents one of the hallmarks of Alzheimer's disease. We employed solid-state nuclear magnetic resonance (NMR) to investigate the structure and dynamics of PHFs formed in vitro by the three-repeat-domain (K19) of protein tau, representing the core of Alzheimer PHFs. While N and C termini of tau monomers in PHFs are highly dynamic and solvent-exposed, the rigid segment consists of three major beta-strands. Combination of through-bond and through-space ssNMR transfer methods with water-edited ((15)N, (13)C) and ((13)C, (13)C) correlation experiments suggests the existence of a fibril core that is largely built by repeat unit R3, flanked by surface-exposed units R1 and R4. Solid-state NMR, circular dichroism, and the fibrillization behavior of a K19 mutant furthermore indicate that electrostatic interactions play a central role in stabilizing the K19 PHFs.     

Semisynthetic and Enzyme‐Mediated Conjugate Preparations Illuminate the Ubiquitination‐Dependent Aggregation of Tau Protein

In the brain of individuals with Alzheimer's disease, the regulatory protein ubiquitin is found conjugated to different lysine residues of tau protein assembled into pathological paired helical filaments. To shed light on the hitherto unexplored ubiquitination‐linked conformational transitions of tau, the availability of in vitro ubiquitin conjugation methods is of primary importance. In our work, we focused on the four‐repeat domain of tau and assembled an enzymatic machinery formed by UBE1, Ubc13, and CHIP enzymes. The enzymatic reaction resulted in monoubiquitination at multiple sites, reminiscent of the ubiquitination pattern observed in vivo. We further exploited chemoselective disulfide coupling reactions to construct three tau regioisomers with site‐specific monoubiquitination. Protein aggregation experiments revealed that the multiple enzyme‐derived products were unable to convert into amyloid fibrils, while the semisynthetic conjugates exhibited diverse capability to form filaments. This study contributes novel insight into the effects of a key post‐translational modification on aberrant protein self‐assembly.     

Semisynthetic and Enzyme-Mediated Conjugate Preparations Illuminate the Ubiquitination-Dependent Aggregation of Tau Protein

In the brain of individuals with Alzheimer's disease, the regulatory protein ubiquitin is found conjugated to different lysine residues of tau protein assembled into pathological paired helical filaments. To shed light on the hitherto unexplored ubiquitination-linked conformational transitions of tau, the availability of in vitro ubiquitin conjugation methods is of primary importance. In our work, we focused on the four-repeat domain of tau and assembled an enzymatic machinery formed by UBE1, Ubc13, and CHIP enzymes. The enzymatic reaction resulted in monoubiquitination at multiple sites, reminiscent of the ubiquitination pattern observed in vivo. We further exploited chemoselective disulfide coupling reactions to construct three tau regioisomers with site-specific monoubiquitination. Protein aggregation experiments revealed that the multiple enzyme-derived products were unable to convert into amyloid fibrils, while the semisynthetic conjugates exhibited diverse capability to form filaments. This study contributes novel insight into the effects of a key post-translational modification on aberrant protein self-assembly.     

Electron bubbles in helium clusters. II. Probing superfluidity

In this paper we present calculations of electron tunneling times from the ground electronic state of excess electron bubbles in ((4)He)(N) clusters (N=6500-10(7), cluster radius R=41.5-478 A), where the equilibrium bubble radius varies in the range R(b)=13.5-17.0 A. For the bubble center located at a radial distance d from the cluster surface, the tunneling transition probability was expressed as A(0)phi(d,R)exp(-betad), where beta approximately 1 A(-1) is the exponential parameter, A(0) is the preexponential factor for the bubble located at the cluster center, and phi(d,R) is a correction factor which accounts for cluster curvature effects. Electron tunneling dynamics is grossly affected by the distinct mode of motion of the electron bubble in the image potential within the cluster, which is dissipative (i.e., tau(D)tau(0)) in superfluid ((4)He)(N) clusters, where tau(D) is the bubble motional damping time (tau(D) approximately 4 x 10(-12) s for normal fluid clusters and tau(D) approximately 10 s for superfluid clusters), while tau(0) approximately 10(-9)-10(-10) s is the bubble oscillatory time. Exceedingly long tunneling lifetimes, which cannot be experimentally observed, are manifested from bubbles damped to the center of the normal fluid cluster, while for superfluid clusters electron tunneling occurs from bubbles located in the vicinity of the initial distance d near the cluster boundary. Model calculations of the cluster size dependence of the electron tunneling time (for a fixed value of d=38-39 A), with lifetimes increasing in the range of 10(-3)-0.3 s for N=10(4)-10(7), account well for the experimental data [M. Farnik and J. P. Toennies, J. Chem. Phys. 118, 4176 (2003)], manifesting cluster curvature effects on electron tunneling dynamics. The minimal cluster size for the dynamic stability of the bubble was estimated to be N=3800, which represents the threshold cluster size for which the excess electron bubble in ((4)He)(N) (-) clusters is amenable to experimental observation.     

Molecular dynamics simulation of [Gd(egta)(H(2)O)](-) in aqueous solution: internal motions of the poly(amino carboxylate) and water ligands, and rotational correlation times

Molecular dynamics simulations of [Gd(egta)(H(2)O)](-) (egta(4-)=3,12-bis(carboxymethyl)-6,9-dioxa-3,12-diazatetradecanedioate(4-)) have been performed without any artificial constraint on the first coordination sphere, such as covalent bonds between the Gd(3+) and the coordination sites. Two new crystallographic structures were determined for this gadolinium chelate and used to start two molecular dynamics simulations. [Gd(egta)(H(2)O)](-) and [Gd(egta)](-) were both observed during the simulations, with a mean volume for the reaction of dissociation [Gd(egta)(H(2)O)](-)-->[Gd(egta)](-)+H(2)O of +7.2 cm(3)mol(-1), which corroborates the previously published experimental value of +10.5 cm(3)mol(-1). Changes in the conformation of the complex with the inversion of several dihedral angles are observed in the simulations independently from the water dissociation. Very fast changes of the third-order rotation axis direction of the Gd(3+) coordination polyhedron (of symmetry D(3h)) are observed during the simulations and are related to the mechanism of electronic relaxation of the complex. Different rotational correlation times (tau(R)) were calculated from the simulations on various observables of the complex. Protons of the inner sphere have different tau(R). The mean tau(R) of the two Gd-HW(HW=hydrogen of water molecule) vectors is 72% lower than tau(R) of the complex, and 75% lower than tau(R) of the vector Gd-OW (OW=oxygen of water molecule). This discrimination of the tumbling rates should be taken into account in future global (17)O NMR, EPR and NMRD (nuclear magnetic relaxation dispersion) data analysis.     

A Long‐Living Bioengineered Neural Tissue Platform to Study Neurodegeneration

The prevalence of dementia and other neurodegenerative diseases continues to rise as age demographics in the population shift, inspiring the development of long‐term tissue culture systems with which to study chronic brain disease. Here, it is investigated whether a 3D bioengineered neural tissue model derived from human induced pluripotent stem cells (hiPSCs) can remain stable and functional for multiple years in culture. Silk‐based scaffolds are seeded with neurons and glial cells derived from hiPSCs supplied by human donors who are either healthy or have been diagnosed with Alzheimer's disease. Cell retention and markers of stress remain stable for over 2 years. Diseased samples display decreased spontaneous electrical activity and a subset displays sporadic‐like indicators of increased pathological β‐amyloid and tau markers characteristic of Alzheimer's disease with concomitant increases in oxidative stress. It can be concluded that the long‐term stability of the platform is suited to study chronic brain disease including neurodegeneration.     

Structural, spectroscopic, and electrochemical properties of a series of high-spin thiolatonickel(II) complexes

A series of high-spin thiolatonickel(II) complexes, [PhTttBu]Ni(SR) (PhTttBu = phenyltris((tert-butylthio)methyl)borate; 2, R = triphenylmethyl; 3, R = pentafluorophenyl; 4, R = phenyl), were synthesized via the reaction of [PhTttBu]Ni(NO3) (1) with thiols (RSH) in the presence of triethylamine. The [PhTttBu]Ni(SR) products were isolated and characterized by various physicochemical measurements including X-ray diffraction analyses. These thiolatonickel(II) complexes have a distorted trigonal pyramidal geometry with somewhat different tau values: 0.80 and 0.90 for two crystalline phases of 2, 0.74 for 3, and 0.69 for 4, where tau is a normalized measure of pyramidalization (tau = 0 for tetrahedron, tau = 1 for trigonal pyramid). The electronic absorption spectra display characteristic sulfur-to-nickel(II) charge transfer (CT) bands at 532 nm (7500 M(-1) cm(-1)) for 2, 510 nm (4800 M(-1) cm(-1)) for 3, and 569 nm (4100 M(-1) cm(-1)) for 4. The cyclic voltammograms show a quasi-reversible redox couple at E1/2 = -1.11 V for 2, and reversible redox couples at E1/2 = -1.03 V for 3 and E1/2 = -1.17 V for 4 (vs Fc+/Fc). Correlation between the tau value and the CT intensity was observed: the strong CT intensity results from the high tau value, which provides for strong orbital overlap (2 > 3 > 4). Additionally, the CT transition energy correlates with the reduction potential: both the CT transition energy and potential decrease in the order 3 > 2 > 4, consistent with the influence of decreasing electron withdrawing abilities, R = pentafluorophenyl > triphenylmethyl > phenyl. The three thiolatonickel complexes exhibit dramatically different thermal stabilities. Complex 4 is the least stable, undergoing decomposition to [kappa2-PhBttBuSPh]Ni(eta2-CH2SBut) (5) via net exchange of Ni-SPh and B-CH2SBut groups.     

Atomic force microscopic imaging of seeded fibril formation and fibril branching by the Alzheimer's disease amyloid-β protein

Background: Amyloid plaques composed of the fibrillar form of the amyloid-β protein (Aβ) are the defining neuropathological feature of Alzheimer's disease (AD). A detailed understanding of the time course of amyloid formation could define steps in disease progression and provide targets for therapeutic intervention. Amyloid fibrils, indistinguishable from those derived from an AD brain, can be produced in vitro using a seeded polymerization mechanism. In its simplest form, this mechanism involves a cooperative transition from monomeric Aβ to the amyloid fibril without the buildup of intermediates. Recently, however, a transient species, the Aβ amyloid protofibril, has been identified. Here, we report studies of Aβ amyloid protofibril and its seeded transition into amyloid fibrils using atomic force microscopy.Results: Seeding of the protofibril-to-fibril transition was observed. Preformed fibrils, but not protofibrils, effectively seeded this transition. The assembly state of Aβ influenced the rate of seeded growth, indicating that protofibrils are fibril assembly precursors. The handedness of the helical surface morphology of fibrils depended on the chirality of Aβ. Finally, branched and partially wound fibrils were observed.Conclusions: The temporal evolution of morphologies suggests that the protofibril-to-fibril transition is nucleation-dependent and that protofibril winding is involved in that transition. Fibril unwinding and branching may be essential for the post-nucleation growth process. The protofibrillar assembly intermediate is a potential target for AD therapeutics aimed at inhibiting amyloid formation and AD diagnostics aimed at detecting presymptomatic disease.     

Photoinduced electron transfer of N-[(3- and 4-diarylamino)phenyl]-1,8-naphthalimide dyads: orbital-orthogonal approach in a short-linked D-A system

The photoinduced electron transfer of a series of meta- and para-linked triphenylamine-naphthalimide dyads, N-{3- and 4-[bis(4-R-substituted phenyl)amino]phenyl}-1,8-naphthalimide, 1m,p (R = H), 2m,p (R = Me), 3m,p (R = OMe), and 4m,p (R = NMe2) was investigated in toluene and DMF. The singlet charge-transfer (CT) states were observed in all cases. The decay rates were found to be faster in DMF (tau = 6.5 ps to 100 ps) than those in toluene (tau = 190 ps to 7 ns). The long-lived triplet CT states were observed in toluene for 3 (ca. 10% contribution, tau = 670 ns for 3m, 240 ns for 3p). No long-lived species were detected in DMF. The decay rates were somewhat faster in the para-isomers than in the meta-isomers in most cases. The photolysis of 5 (p-phenylene extended analogue of 3, R = OMe) gave a singlet CT state and a locally excited triplet state on the naphthalimide chromophore.     

Pentameric Thiophene‐Based Ligands that Spectrally Discriminate Amyloid‐β and Tau Aggregates Display Distinct Solvatochromism and Viscosity‐Induced Spectral Shifts

A wide range of neurodegenerative diseases are characterized by the deposition of multiple protein aggregates. Ligands for molecular characterization and discrimination of these pathological hallmarks are thus important for understanding their potential role in pathogenesis as well as for clinical diagnosis of the disease. In this regard, luminescent conjugated oligothiophenes (LCOs) have proven useful for spectral discrimination of amyloid‐beta (Aβ) and tau neurofibrillary tangles (NFTs), two of the pathological hallmarks associated with Alzheimer’s disease. Herein, the solvatochromism of a library of anionic pentameric thiophene‐based ligands, as well as their ability to spectrally discriminate Aβ and tau aggregates, were investigated. Overall, the results from this study identified distinct solvatochromic and viscosity‐dependent behavior of thiophene‐based ligands that can be applied as indices to direct the chemical design of improved LCOs for spectral separation of Aβ and tau aggregates in brain tissue sections. The results also suggest that the observed spectral transitions of the ligands are due to their ability to conform by induced fit to specific microenvironments within the binding interface of each particular protein aggregate. We foresee that these findings might aid in the chemical design of thiophene‐based ligands that are increasingly selective for distinct disease‐associated protein aggregates.     

Spectroscopic characteristics of Sm3+-doped alkali fluorophosphate glasses

Optical absorption, luminescence and lifetime measurements of Sm(3+)-doped alkali fluorophosphate glasses with molar compositions of 50(NaPO(3))(6)+10TeO(2)+20AlF(3)+19RF+1Sm(2)O(3) (R=Li, Na and K) are described. The variation of optical properties with glass composition plays a dominant role in the determination of efficient laser materials. From the experimental oscillator strengths of f-f electric dipole transitions, the phenomenological Judd-Ofelt parameters have been evaluated and are used to evaluate radiative parameters such as radiative transition probabilities (A(R)), branching ratios (beta(R)), lifetimes (tau(R)) and integrated absorption cross-section (sigma(a)) for various excited levels. The predicted values of tau(R) and beta(R) from the (4)G(5/2) excited level to its lower levels are compared with the experimentally measured values. Stimulated emission cross-sections (sigma(e)) were also determined for (4)G(5/2)-->(6)H(J) (J=5/2, 7/2, 9/2 and 11/2) emission transitions. From the emission transitions of Sm(3+) in these alkali tellurofluorophosphate glasses certain potential laser transitions have been identified.     

Forced dissociation of a biomolecular complex under periodic and correlated random forcing

The dissociation of a biomolecular complex under the action of periodic and correlated random forcing is studied theoretically. The former is characterized by the period tau p and the latter by the correlation time tau r. The rupture rates are calculated by overdamped Langevin dynamics and three distinct regimes are identified for both cases by comparison to local relaxation time tau R and bond lifetime T. For periodic forcing, the adiabatic approximation cannot be applied in the regime tau p相似文献   

Spectroscopic properties of Dy(3+) ions in NaF-B(2)O(3)-Al(2)O(3) glasses

This paper reports the optical properties of Dy(3+) in sodium fluoroborate glasses of the type XNaF.(89-X)B(2)O(3).10 Al(2)O(3).1Dy(2)O(3) (where X=8, 12, 16, 20 and 24). Judd-Ofelt intensity parameters (Omega(2), Omega(4), Omega(6)) are derived from the absorption spectra. The Judd-Ofelt theory has been applied to interpret the local environment of Dy(3+) ions and bond covalency of RE-O bond. These parameters have been used to calculate radiative transition probabilities (A(rad)), lifetimes (tau(R)) and branching ratios (beta(R)) for the excited level (4)F(9/2). The predicted values of tau(R) are compared with the measured values for (4)F(9/2) level for five glass compositions (Glass (A-E)). The stimulated emission cross-section sigma(lambda(P)) are also evaluated for the (4)F(9/2)-->(6)H(J) (J=11/2, 13/2, and 15/2) transitions.     

Distinct Spacing Between Anionic Groups: An Essential Chemical Determinant for Achieving Thiophene‐Based Ligands to Distinguish β‐Amyloid or Tau Polymorphic Aggregates

The accumulation of protein aggregates is associated with many devastating neurodegenerative diseases and the existence of distinct aggregated morphotypes has been suggested to explain the heterogeneous phenotype reported for these diseases. Thus, the development of molecular probes able to distinguish such morphotypes is essential. We report an anionic tetrameric oligothiophene compound that can be utilized for spectral assignment of different morphotypes of β‐amyloid or tau aggregates present in transgenic mice at distinct ages. The ability of the ligand to spectrally distinguish between the aggregated morphotypes was reduced when the spacing between the anionic substituents along the conjugated thiophene backbone was altered, which verified that specific molecular interactions between the ligand and the protein aggregate are necessary to detect aggregate polymorphism. Our findings provide the structural and functional basis for the development of new fluorescent ligands that can distinguish between different morphotypes of protein aggregates.     

Macroscopic deformation of a substrate as a new method for controlling geometry and plasmon-resonant properties of highly ordered planar ensembles of metal nanoparticles

A new approach to creating highly ordered two-dimensional ensembles of nanoparticles with variable geometric parameters is proposed. It combines diblock copolymer micellar lithography and controlled deformation of a polymer substrate. The key feature of the approach is the formation of a monolayer of hexagonally packed metal precursor-containing micelles of an amphiphilic diblock copolymer on the surface of an isotropically stretched polymer plate. The average distance between micelle centers is 140 nm. Subsequent thermal treatment (or isotropic stretching) of the sample results in the shrinkage (or elongation) of the substrate, which enables one to vary the distance between micelle centers in a range of 80–200 nm while retaining hexagonal packing of the micelles in the monolayer. At the final stage, ensembles of hexagonally ordered gold nanoparticles are obtained by exposing the micellar films to air plasma. It is demonstrated that gold nanoparticles in these ensembles can be enlarged by seeded growth. The systematic study of the plasmon-resonant properties of the resulting ensembles shows that the gradual increase in the distance between 35-nm gold particles from 80 to 200 nm leads to an unexpected nonmonotonic shift of the maximum of localized surface plasmon resonance, which is, from our point of view, caused by the high degree of organization of nanoparticles on the substrate.     

Structural insights into the polymorphism of amyloid-like fibrils formed by region 20-29 of amylin revealed by solid-state NMR and X-ray fiber diffraction

Many unrelated proteins and peptides can assemble into amyloid or amyloid-like nanostructures, all of which share the cross-beta motif of repeat arrays of beta-strands hydrogen-bonded along the fibril axis. Yet, paradoxically, structurally polymorphic fibrils may derive from the same initial polypeptide sequence. Here, solid-state nuclear magnetic resonance (SSNMR) analysis of amyloid-like fibrils of the peptide hIAPP 20-29, corresponding to the region S (20)NNFGAILSS (29) of the human islet amyloid polypeptide amylin, reveals that the peptide assembles into two amyloid-like forms, (1) and (2), which have distinct structures at the molecular level. Rotational resonance SSNMR measurements of (13)C dipolar couplings between backbone F23 and I26 of hIAPP 20-29 fibrils are consistent with form (1) having parallel beta-strands and form (2) having antiparallel strands within the beta-sheet layers of the protofilament units. Seeding hIAPP 20-29 with structurally homogeneous fibrils from a 30-residue amylin fragment (hIAPP 8-37) produces morphologically homogeneous fibrils with similar NMR properties to form (1). A model for the architecture of the seeded fibrils is presented, based on the analysis of X-ray fiber diffraction data, combined with an extensive range of SSNMR constraints including chemical shifts, torsional angles, and interatomic distances. The model features a cross-beta spine comprising two beta-sheets with an interface defined by residues F23, A25, and L27, which form a hydrophobic zipper. We suggest that the energies of formation for fibril form containing antiparallel and parallel beta-strands are similar when both configurations can be stabilized by a core of hydrophobic contacts, which has implications for the relationship between amino acid sequence and amyloid polymorphism in general.     

Chemical Imaging of RNA-Tau Amyloid Fibrils at the Nanoscale Using Tip-Enhanced Raman Spectroscopy

In the presence of cofactors, tau protein can form amyloid deposits in the brain which are implicated in many neurodegenerative disorders. Heparin, lipids, and RNA are used to recreate tau aggregates in vitro from recombinant protein. However, the mechanism of interaction of these cofactors and the interactions between cofactors and tau are poorly understood. Herein, we use tip-enhanced Raman spectroscopy (TERS) to visualize the spatial distribution of adenine, protein secondary structure, and amino acids (arginine, lysine and histidine) in single polyadenosine (polyA)-induced tau fibrils with nanoscale spatial resolution (<10–20 nm). Based on reference unenhanced and surface-enhanced Raman spectra, we show that the polyA anionic cofactor is incorporated in the fibril structure and seems to be superficial to the β-sheet core, but nonetheless enveloped within the random-coiled fuzzy coat. TERS images also prove the colocalization of positively charged arginine, lysine, and histidine amino acids and negatively charged polyA, which constitutes an important step forward to better comprehend the action of RNA cofactors in the mechanism of formation of toxic tau fibrils. TERS appears as a powerful technique for the identification of cofactors in individual tau fibrils and their mode of interaction.     

Nanoscopic optical sensors based on functional supramolecular hybrid materials

This review highlights how the combination of supramolecular principles and nanoscopic solid structures enables the design of new hybrid sensing ensembles with improved sensitivity and/or selectivity and for the targeting of analytes for which selectivity is hard to achieve by conventional methods. Such ideas are bridging the gap between molecules, materials sciences and nanotechnology. Relevant examples will be detailed, taking into account functional aspects such as (1) enhanced coordination of functionalized solids, (2) enhanced signalling through preorganization, (3) signalling by assembly–disassembly of nanoscopic objects, (4) biomimetic probes utilizing discrimination by polarity and size and (5) distinct switching and gating protocols. These strategies are opening new prospects for sensor research and signalling paradigms at the frontier between nanotechnology, smart materials and supramolecular chemistry.     

Probing charge separation in structurally different C60/exTTF ensembles

The scope of the present work is to highlight the effects stemming from different C60/exTTF linkages (exTTF = 9,10-bis(1,3-dithiol-2-ylidene)-9,10-dihydroanthracene)-either via an anthracene unit or a dithiole ring. Particular emphasis is placed on photoinduced electron-transfer features. Therefore, we devised a new series of C60-exTTF ensembles, synthesized via 1,3-dipolar cycloaddition and Diels-Alder cycloaddition reactions, in which exTTF units are separated from C60 by two single bonds (3a-c, 4), one vinylene unit (5a), or two vinylene units (5b). The cyclic voltammetry reveals an amphoteric redox behavior with remarkably strong electron-donor ability of the trimethyl-substituted exTTF moiety in 4 and 5a,b. Steady-state and time-resolved photolytic techniques show that the fullerene singlet excited state in (3a-c, 4, and 5a,b) is subject to a rapid electron-transfer quenching. The resulting charge-separated states, that is C60*(-)-exTTF*+, were identified by transient absorption spectroscopy. We determined radical pair lifetimes of the order of 200 ns in benzonitrile. This suggests (i) that the positive charge of the exTTF*+ is delocalized over the entire donor rather than localized on one of the 1,3-dithiole rings and (ii) that linking exTTF via the anthracene or 1,3-dithiole ring has no appreciable influence. Increasing the donor-acceptor separation via implementing one or two vinylene units as spacers led to improved radical pair lifetimes (5a: tau = 725 ns; 5b: tau = 1465 ns).     

Polymer translocation through a nanopore: a two-dimensional Monte Carlo study

We investigate the problem of polymer translocation through a nanopore in the absence of an external driving force. To this end, we use the two-dimensional fluctuating bond model with single-segment Monte Carlo moves. To overcome the entropic barrier without artificial restrictions, we consider a polymer which is initially placed in the middle of the pore and study the escape time tau required for the polymer to completely exit the pore on either end. We find numerically that tau scales with the chain length N as tau approximately N(1+2nu), where nu is the Flory exponent. This is the same scaling as predicted for the translocation time of a polymer which passes through the nanopore in one direction only. We examine the interplay between the pore length L and the radius of gyration R(g). For LR(g), we find tau approximately N. In addition, we numerically find the scaling function describing crossover between short and long pores. We also show that tau has a minimum as a function of L for longer chains when the radius of gyration along the pore direction R( parallel) approximately L. Finally, we demonstrate that the stiffness of the polymer does not change the scaling behavior of translocation dynamics for single-segment dynamics.