Hypertrophic cardiomyopathy mutations Y115H and E497D disrupt the folded-back state of human beta-cardiac myosin allosterically.

At the molecular level, clinical hypercontractility associated with many hypertrophic cardiomyopathy (HCM)-causing mutations in beta-cardiac myosin appears to be driven by their disruptive effect on the energy-conserving, folded-back, super relaxed (SRX) OFF-state of myosin. A pathological increase in force production results from release of heads from this OFF-state, which results in an increase in the number of heads free to interact with actin and produce force. Pathogenic mutations in myosin can conceivably disrupt the OFF-state by (1) directly affecting the intramolecular interfaces stabilizing the folded-back state, or (2) allosterically destabilizing the folded-back state via disruption of diverse conformational states of the myosin motor along its chemomechanical cycle. However, very little is understood about the mutations that fall in the latter group. Here, using recombinant human beta-cardiac myosin, we analysed the biomechanical properties of two such HCM-causing mutations, Y115H (in the transducer) and E497D (in the relay helix), neither of which falls in the regions that interact to stabilize the myosin folded-back state. We find these mutations have diverse effects on the contractility parameters of myosin, yet the primary hypercontractile change in both cases is the destabilization of the OFF-state of myosin and increased availability of active myosin heads for actin-binding. Experimental data and molecular dynamics simulations indicate that these mutations likely destabilize the pre-powerstroke state of myosin, the conformation the motor adopts in the inactive folded-back state. We propose that destabilization of the folded-back state of myosin, directly and/or allosterically, is the molecular basis of hypercontractility in HCM in a far greater number of pathogenic mutations than currently thought.

Hypertrophic cardiomyopathy mutations in the pliant and light chain-binding regions of the lever arm of human ß-cardiac myosin have divergent effects on myosin function.

Mutations in the lever arm of ß-cardiac myosin are a frequent cause of hypertrophic cardiomyopathy, a disease characterized by hypercontractility and eventual hypertrophy of the left ventricle. Here, we studied five such mutations: three in the pliant region of the lever arm (D778V, L781P, and S782N) and two in the light chain-binding region (A797T and F834L). We investigated their effects on both motor function and myosin subfragment 2 (S2) tail-based autoinhibition. The pliant region mutations had varying effects on the motor function of a myosin construct lacking the S2 tail: overall, D778V increased power output, L781P reduced power output, and S782N had little effect on power output, while all three reduced the external force sensitivity of the actin detachment rate. With a myosin containing the motor domain and the proximal S2 tail, the pliant region mutations also attenuated autoinhibition in the presence of filamentous actin but had no impact in the absence of actin. By contrast, the light chain-binding region mutations had little effect on motor activity but produced marked reductions in autoinhibition in both the presence and absence of actin. Thus, mutations in the lever arm of ß-cardiac myosin have divergent allosteric effects on myosin function, depending on whether they are in the pliant or light chain-binding regions.

Coexisting Ordered and Disordered Membrane Phases Have Distinct Modes of Interaction with Disease-Associated Oligomers.

Ordered membrane domains are thought to influence the attachment and insertion of toxic amyloid oligomers, and consequently, their toxicity. However, if and how the molecular aspects of this interaction depend on the membrane order is poorly understood. Here we measure the affinity, location, and degree of insertion of the small oligomers of hIAPP (human Islet Amyloid Polypeptide, associated with Type II diabetes) at near-physiological concentrations to adjacent domains of a biphasic lipid bilayer. Using simultaneous atomic force, confocal and fluorescence lifetime microscopy (AFM-FLIM), we find that hIAPP oligomers have a nearly 8-fold higher affinity to the disordered domains over the ordered domains. To probe whether this difference indicates different modes of interaction, we measure the change of lifetime of peptide-attached fluorescent labels induced by soluble fluorescence quenchers and also measure the kinetics of localized photobleaching. We find that in the raft-like ordered domains, the oligomers primarily lie on the aqueous interface with limited membrane penetration. However, in the neighboring disordered domains, their C-termini penetrate deeper into the lipid bilayer. We conclude that local membrane order determines not only the affinity but also the mode of interaction of amyloid oligomers, which may have significant implications for disease mechanisms.

Synapse cell optimization and back-propagation algorithm implementation in a domain wall synapse based crossbar neural network for scalable on-chip learning.

On-chip learning in spin orbit torque driven domain wall synapse based crossbar fully connected neural network (FCNN) has been shown to be extremely efficient in terms of speed and energy, when compared to training on a conventional computing unit or even on a crossbar FCNN based on other non-volatile memory devices. However there are issues with respect to scalability of the on-chip learning scheme in the domain wall synapse based FCNN. Unless the scheme is scalable, it will not be competitive with respect to training a neural network on a conventional computing unit for real applications. In this paper, we have proposed a modification in the standard gradient descent algorithm, used for training such FCNN, by including appropriate thresholding units. This leads to optimization of the synapse cell at each intersection of the crossbars and makes the system scalable. In order for the system to approximate a wide range of functions for data classification, hidden layers must be present and the backpropagation algorithm (extension of gradient descent algorithm for multi-layered FCNN) for training must be implemented on hardware. We have carried this out in this paper by employing an extra crossbar. Through a combination of micromagnetic simulations and SPICE circuit simulations, we hence show highly improved accuracy for domain wall syanpse based FCNN with a hidden layer compared to that without a hidden layer for different machine learning datasets.

Resolving Single-Nanoconstruct Dynamics during Targeting and Nontargeting Live-Cell Membrane Interactions.

This paper describes how differences in the dynamics of targeting and nontargeting constructs can provide information on nanoparticle (NP)-cell interactions. We probed translational and rotational dynamics of functionalized Au nanostar (AuNS) nanoconstructs interacting with cells in serum-containing medium. We found that AuNS with targeting ligands had a larger dynamical footprint and faster rotational speed on cell membranes expressing human epidermal growth factor receptor 2 (HER-2) receptors compared to that of AuNS with nontargeting ligands. Targeting and nontargeting nanoconstructs displayed distinct membrane dynamics despite their similar protein adsorption profiles, which suggests that targeted interactions are preserved even in the presence of a protein corona. The high sensitivity of single-NP dynamics can be used to compare different nanoconstruct properties (such as NP size, shape, and surface chemistry) to improve their design as delivery vehicles.

Lattice-Resonance Metalenses for Fully Reconfigurable Imaging.

This paper describes a reconfigurable metalens system that can image at visible wavelengths based on arrays of coupled plasmonic nanoparticles. These lenses manipulated the wavefront and focused light by exciting surface lattice resonances that were tuned by patterned polymer blocks on single-particle sites. Predictive design of the dielectric nanoblocks was performed using an evolutionary algorithm to create a range of three-dimensional focusing responses. For scalability, we demonstrated a simple technique for erasing and writing the polymer nanostructures on the metal nanoparticle arrays in a single step using solvent-assisted nanoscale embossing. This reconfigurable materials platform enables tunable focusing with diffraction-limited resolution and offers prospects for highly adaptive, compact imaging.

Wavelength-Dependent Differential Interference Contrast Inversion of Anisotropic Gold Nanoparticles.

Gold nanorods are promising nanoparticle-orientation sensors because they exhibit wavelength and angle-dependent optical patterns in their differential interference contrast (DIC) microscopy images. In this paper, we report a finite-difference time-domain method to simulate DIC images using nanorods as model probes. First, we created a DIC image library of nanorods as a function of imaging wavelength and rotation angle that showed good agreement with experimental results. Second, we used this simulation tool to explain why the patterns inverted from bright to dark when the imaging wavelength increased from below to above the plasmon resonance of the nanorod. We found that this intensity inversion resulted from reversal in electric field direction depending on wavelength relative to the nanorod plasmon resonance. Finally, we showed that this DIC contrast inversion is a general phenomenon by measuring and simulating DIC images from gold nanorods of different sizes and gold nanostars.

Major Reaction Coordinates Linking Transient Amyloid-ß Oligomers to Fibrils Measured at Atomic Level.

The structural underpinnings for the higher toxicity of the oligomeric intermediates of amyloidogenic peptides, compared to the mature fibrils, remain unknown at present. The transient nature and heterogeneity of the oligomers make it difficult to follow their structure. Here, using vibrational and solid-state nuclear magnetic resonance spectroscopy, and molecular dynamics simulations, we show that freely aggregating Aß40 oligomers in physiological solutions have an intramolecular antiparallel configuration that is distinct from the intermolecular parallel ß-sheet structure observed in mature fibrils. The intramolecular hydrogen-bonding network flips nearly 90°, and the two ß-strands of each monomeric unit move apart, to give rise to the well-known intermolecular in-register parallel ß-sheet structure in the mature fibrils. Solid-state nuclear magnetic resonance distance measurements capture the interstrand separation within monomer units during the transition from the oligomer to the fibril form. We further find that the D23-K28 salt-bridge, a major feature of the Aß40 fibrils and a focal point of mutations linked to early onset Alzheimer's disease, is not detectable in the small oligomers. Molecular dynamics simulations capture the correlation between changes in the D23-K28 distance and the flipping of the monomer secondary structure between antiparallel and parallel ß-sheet architectures. Overall, we propose interstrand separation and salt-bridge formation as key reaction coordinates describing the structural transition of the small Aß40 oligomers to fibrils.

Curcumin Dictates Divergent Fates for the Central Salt Bridges in Amyloid-ß40 and Amyloid-ß42.

There are three specific regions in the Amyloid beta (Aß) peptide sequence where variations cause enhanced toxicity in Alzheimer's disease: the N-terminus, the central salt bridge, and the C-terminus. Here, we investigate if there is a close conformational connection between these three regions, which may suggest a concerted mechanism of toxicity. We measure the effects of Zn2+ and curcumin on Aß40, and compare these with their previously reported effects on Aß42. Aß42 and Aß40 differ only near the C-terminus, where curcumin interacts, while Zn2+ interacts near the N-terminus. Therefore, this comparison should help us differentiate the effect of modulating the C- and the N-termini. We find that curcumin allows fibril-like structures containing the salt bridge to emerge in the mature Aß40 aggregates, but not in Aß42. In contrast, we find no difference in the effects of Zn+2 on Aß40 and Aß42. In the presence of Zn+2, both of these fail to form proper fibrils, and the salt bridge remains disrupted. These results indicate that modulations of the Aß termini can determine the fate of a salt bridge far away in the sequence, and this has significant consequences for Aß toxicity. We also infer that small molecules can alter oligomer-induced toxicity by modulating the aggregation pathway, without substantially changing the final product of aggregation.

A Detailed Analysis of the Morphology of Fibrils of Selectively Mutated Amyloid ß (1-40).

A small library of rationally designed amyloid ß [Aß(1-40)] peptide variants is generated, and the morphology of their fibrils is studied. In these molecules, the structurally important hydrophobic contact between phenylalanine 19 (F19) and leucine 34 (L34) is systematically mutated to introduce defined physical forces to act as specific internal constraints on amyloid formation. This Aß(1-40) peptide library is used to study the fibril morphology of these variants by employing a comprehensive set of biophysical techniques including solution and solid-state NMR spectroscopy, AFM, fluorescence correlation spectroscopy, and XRD. Overall, the findings demonstrate that the introduction of significant local physical perturbations of a crucial early folding contact of Aß(1-40) only results in minor alterations of the fibrillar morphology. The thermodynamically stable structure of mature Aß fibrils proves to be relatively robust against the introduction of significantly altered molecular interaction patterns due to point mutations. This underlines that amyloid fibril formation is a highly generic process in protein misfolding that results in the formation of the thermodynamically most stable cross-ß structure.

Steric Crowding of the Turn Region Alters the Tertiary Fold of Amyloid-ß18-35 and Makes It Soluble.

Aß self-assembles into parallel cross-ß fibrillar aggregates, which is associated with Alzheimer's disease pathology. A central hairpin turn around residues 23-29 is a defining characteristic of Aß in its aggregated state. Major biophysical properties of Aß, including this turn, remain unaltered in the central fragment Aß18-35. Here, we synthesize a single deletion mutant, ΔG25, with the aim of sterically hindering the hairpin turn in Aß18-35. We find that the solubility of the peptide goes up by more than 20-fold. Although some oligomeric structures do form, solution state NMR spectroscopy shows that they have mostly random coil conformations. Fibrils ultimately form at a much higher concentration but have widths approximately twice that of Aß18-35, suggesting an opening of the hairpin bend. Surprisingly, two-dimensional solid state NMR shows that the contact between Phe(19) and Leu(34) residues, observed in full-length Aß and Aß18-35, is still intact in these fibrils. This is possible if the monomers in the fibril are arranged in an antiparallel ß-sheet conformation. Indeed, IR measurements, supported by tyrosine cross-linking experiments, provide a characteristic signature of the antiparallel ß-sheet. We conclude that the self-assembly of Aß is critically dependent on the hairpin turn and on the contact between the Phe(19) and Leu(34) regions, making them potentially sensitive targets for Alzheimer's therapeutics. Our results show the importance of specific conformations in an aggregation process thought to be primarily driven by nonspecific hydrophobic interactions.

Cell-Membrane-Mimicking Lipid-Coated Nanoparticles Confer Raman Enhancement to Membrane Proteins and Reveal Membrane-Attached Amyloid-ß Conformation.

Identifying the structures of membrane bound proteins is critical to understanding their function in healthy and diseased states. We introduce a surface enhanced Raman spectroscopy technique which can determine the conformation of membrane-bound proteins, at low micromolar concentrations, and also in the presence of a substantial membrane-free fraction. Unlike conventional surface enhanced Raman spectroscopy, our approach does not require immobilization of molecules, as it uses spontaneous binding of proteins to lipid bilayer-encapsulated Ag nanoparticles. We apply this technique to probe membrane-attached oligomers of Amyloid-ß40 (Aß40), whose conformation is keenly sought in the context of Alzheimer's disease. Isotope-shifts in the Raman spectra help us obtain secondary structure information at the level of individual residues. Our results show the presence of a ß-turn, flanked by two ß-sheet regions. We use solid-state NMR data to confirm the presence of the ß-sheets in these regions. In the membrane-attached oligomer, we find a strongly contrasting and near-orthogonal orientation of the backbone H-bonds compared to what is found in the mature, less-toxic Aß fibrils. Significantly, this allows a "porin" like ß-barrel structure, providing a structural basis for proposed mechanisms of Aß oligomer toxicity.

Switching of perpendicularly polarized nanomagnets with spin orbit torque without an external magnetic field by engineering a tilted anisotropy.

Spin orbit torque (SOT) provides an efficient way to significantly reduce the current required for switching nanomagnets. However, SOT generated by an in-plane current cannot deterministically switch a perpendicularly polarized magnet due to symmetry reasons. On the other hand, perpendicularly polarized magnets are preferred over in-plane magnets for high-density data storage applications due to their significantly larger thermal stability in ultrascaled dimensions. Here, we show that it is possible to switch a perpendicularly polarized magnet by SOT without needing an external magnetic field. This is accomplished by engineering an anisotropy in the magnets such that the magnetic easy axis slightly tilts away from the direction, normal to the film plane. Such a tilted anisotropy breaks the symmetry of the problem and makes it possible to switch the magnet deterministically. Using a simple Ta/CoFeB/MgO/Ta heterostructure, we demonstrate reversible switching of the magnetization by reversing the polarity of the applied current. This demonstration presents a previously unidentified approach for controlling nanomagnets with SOT.

Deterministic Domain Wall Motion Orthogonal To Current Flow Due To Spin Orbit Torque.

Spin-polarized electrons can move a ferromagnetic domain wall through the transfer of spin angular momentum when current flows in a magnetic nanowire. Such current induced control of a domain wall is of significant interest due to its potential application for low power ultra high-density data storage. In previous reports, it has been observed that the motion of the domain wall always happens parallel to the current flow - either in the same or opposite direction depending on the specific nature of the interaction. In contrast, here we demonstrate deterministic control of a ferromagnetic domain wall orthogonal to current flow by exploiting the spin orbit torque in a perpendicularly polarized Ta/CoFeB/MgO heterostructure in presence of an in-plane magnetic field. Reversing the polarity of either the current flow or the in-plane field is found to reverse the direction of the domain wall motion. Notably, such orthogonal motion with respect to current flow is not possible from traditional spin transfer torque driven domain wall propagation even in presence of an external magnetic field. Therefore the domain wall motion happens purely due to spin orbit torque. These results represent a completely new degree of freedom in current induced control of a ferromagnetic domain wall.

An early folding contact between Phe19 and Leu34 is critical for amyloid-ß oligomer toxicity.

Small hydrophobic oligomers of aggregation-prone proteins are thought to be generically toxic. Here we examine this view by perturbing an early folding contact between Phe19 and Leu34 formed during the aggregation of Alzheimer's amyloid-ß (Aß40) peptide. We find that even conservative single mutations altering this interaction can abolish Aß40 toxicity. Significantly, the mutants are not distinguishable either by the oligomers size or by the end-state fibrillar structure from the wild type Aß40. We trace the change in their toxicity to a drastic lowering of membrane affinity. Therefore, nonlocal folding contacts play a key role in steering the oligomeric intermediates through specific conformations with very different properties and toxicity levels. Our results suggest that engineering the folding energy landscape may provide an alternative route to Alzheimer therapeutics.

Rapid, cell-free assay for membrane-active forms of amyloid-ß.

Small oligomers of amyloid beta (Aß) are suspected to be the key to Alzheimer's disease (AD). However, identifying these toxic species in the background of other similar but nontoxic Aß aggregates has remained a challenge. Recent studies indicate that Aß undergoes a global structural transition in an early step of aggregation. This transition is marked by a strong increase in its affinity for cell membranes, which suggests that the resultant oligomers could be the key to Aß toxicity. Here we use this increased membrane affinity to develop a rapid, quantitative, cell-free assay for these bioactive oligomers. It uses fluorescence correlation spectroscopy of fluorescently labeled Aß and requires only 30 s of measurement time. We also describe a simpler (though less rapid) assay based on the same principles, which uses a dialysis step followed by conventional fluorescence spectroscopy. Our results potentially provide a much-needed high-throughput assay for AD drug development.

Significant structural differences between transient amyloid-ß oligomers and less-toxic fibrils in regions known to harbor familial Alzheimer's mutations.

Small oligomers of the amyloidâ€…ß (Aß) peptide, rather than the monomers or the fibrils, are suspected to initiate Alzheimer's disease (AD). However, their low concentration and transient nature under physiological conditions have made structural investigations difficult. A method for addressing such problems has been developed by combining rapid fluorescence techniques with slower two-dimensional solid-state NMR methods. The smallest Aß40 oligomers that demonstrate a potential sign of toxicity, namely, an enhanced affinity for cell membranes, were thus probed. The two hydrophobic regions (residues 10-21 and 30-40) have already attained the conformation that is observed in the fibrils. However, the turn region (residues 22-29) and the N-terminal tail (residues 1-9) are strikingly different. Notably, ten of eleven known Aß mutants that are linked to familial AD map to these two regions. Our results provide potential structural cues for AD therapeutics and also suggest a general method for determining transient protein structures.

Curcumin alters the salt bridge-containing turn region in amyloid ß(1-42) aggregates.

Amyloid ß (Aß) fibrillar deposits in the brain are a hallmark of Alzheimer disease (AD). Curcumin, a common ingredient of Asian spices, is known to disrupt Aß fibril formation and to reduce AD pathology in mouse models. Understanding the structural changes induced by curcumin can potentially lead to AD pharmaceutical agents with inherent bio-compatibility. Here, we use solid-state NMR spectroscopy to investigate the structural modifications of amyloid ß(1-42) (Aß42) aggregates induced by curcumin. We find that curcumin induces major structural changes in the Asp-23-Lys-28 salt bridge region and near the C terminus. Electron microscopy shows that the Aß42 fibrils are disrupted by curcumin. Surprisingly, some of these alterations are similar to those reported for Zn(2+) ions, another agent known to disrupt the fibrils and alter Aß42 toxicity. Our results suggest the existence of a structurally related family of quasi-fibrillar conformers of Aß42, which is stabilized both by curcumin and by Zn(2+.)

Spin Hall effect clocking of nanomagnetic logic without a magnetic field.

Spin-based computing schemes could enable new functionalities beyond those of charge-based approaches. Examples include nanomagnetic logic, where information can be processed using dipole coupled nanomagnets, as demonstrated by multi-bit computing gates. One fundamental benefit of using magnets is the possibility of a significant reduction in the energy per bit compared with conventional transistors. However, so far, practical implementations of nanomagnetic logic have been limited by the necessity to apply a magnetic field for clocking. Although the energy associated with magnetic switching itself could be very small, the energy necessary to generate the magnetic field renders the overall logic scheme uncompetitive when compared with complementary metal-oxide-semiconductor (CMOS) counterparts. Here, we demonstrate a nanomagnetic logic scheme at room temperature where the necessity for using a magnetic field clock can be completely removed by using spin-orbit torques. We construct a chain of three perpendicularly polarized CoFeB nanomagnets on top of a tantalum wire and show that an unpolarized current flowing through the wire can 'clock' the perpendicular magnetization to a metastable state. An input magnet can then drive the nanomagnetic chain deterministically to one of two dipole-coupled states, '2 up 1 down' or '2 down 1 up', depending on its own polarization. Thus, information can flow along the chain, dictated by the input magnet and clocked solely by a charge current in tantalum, without any magnetic field. A three to four order of magnitude reduction in energy dissipation is expected for our scheme when compared with state-of-the-art nanomagnetic logic.

pH changes the aggregation propensity of amyloid-ß without altering the monomer conformation.

Decoupling conformational changes from aggregation will help us understand amyloids better. Here we attach Alzheimer's amyloid-ß(1-40) monomers to silver nanoparticles, preventing their aggregation, and study their conformation under aggregation-favoring conditions using SERS. Surprisingly, the α-helical character of the peptide remains unchanged between pH 10.5 and 5.5, while the solubility changes >100×. Amyloid aggregation can therefore start without significant conformational changes.