Protein structural and surface water rearrangement constitute major events in the earliest aggregation stages of tau

Protein aggregation plays a critical role in the pathogenesis of neurodegenerative diseases, and the mechanism of its progression is poorly understood. Here, we examine the structural and dynamic characteristics of transiently evolving protein aggregates under ambient conditions by directly probing protein surface water diffusivity, local protein segment dynamics, and interprotein packing as a function of aggregation time, along the third repeat domain and C terminus of Δtau187 spanning residues 255–441 of the longest isoform of human tau. These measurements were achieved with a set of highly sensitive magnetic resonance tools that rely on site-specific electron spin labeling of Δtau187. Within minutes of initiated aggregation, the majority of Δtau187 that is initially homogeneously hydrated undergoes structural transformations to form partially structured aggregation intermediates. This is reflected in the dispersion of surface water dynamics that is distinct around the third repeat domain, found to be embedded in an intertau interface, from that of the solvent-exposed C terminus. Over the course of hours and in a rate-limiting process, a majority of these aggregation intermediates proceed to convert into stable β-sheet structured species and maintain their stacking order without exchanging their subunits. The population of β-sheet structured species is >5% within 5 min of aggregation and gradually grows to 50–70% within the early stages of fibril formation, while they mostly anneal block-wisely to form elongated fibrils. Our findings suggest that the formation of dynamic aggregation intermediates constitutes a major event occurring in the earliest stages of tau aggregation that precedes, and likely facilitates, fibril formation and growth.The question of whether there is a unified mechanism for amyloid formation that contributes to the progression of neurodegenerative diseases, including Alzheimer’s disease (AD), is a subject of intense debate (1). This view is fueled by the observation that a wide range of amyloidogenic proteins or peptides with different primary sequences can ultimately aggregate into highly structured amyloid fibrils with similar morphologies (2). It has been recognized that a common feature of neurodegenerative diseases is associated with the accumulation and deposits of misfolded proteins that affect various cell signaling processes (3). Among them, the pathological form of a microtubule-associated protein, tau, can dissociate from microtubules and aggregate, resulting in the deposition of insoluble neurofibrillary tangles in neuronal cells (3, 4). Several lines of evidence suggest that small aggregated protein intermediates, that may constitute soluble oligomers and precede the formation of highly structured fibrils, are primary toxic species contributing to the early onset of neurodegenerative diseases (4–6). For example, the presence of granular tau oligomers with prefilamentous structures in transgenic mice was found to correlate with the earliest sign of cognitive decline and memory loss (6). Although early aggregation intermediates formed before fibrils may represent effective targets for common therapeutic intervention, knowledge about their structure and properties is only now emerging. The studies of early intermediates—many of which currently focus on amyloid-β, and comparably few on tau oligomers—are challenging, given the transient nature and complex equilibria involving monomer and oligomer species (4, 7, 8). Recently, several computational studies have identified structural and dynamic features of intermediate aggregates at the molecular level (9, 10), as well as possible driving forces at different aggregation stages (10, 11). Characterization of freeze-trapped amylospheroids by solid-state NMR showed that intermediate structures of amyloid-β aggregates are composed of single conformers containing parallel β-sheets (12). Still, direct experimental observations of transient aggregation and/or folding intermediates remain sparse (5, 12–15).We have previously established a broadly applicable spectroscopic method, Overhauser dynamic nuclear polarization-enhanced NMR relaxometry (ODNP) (16, 17), to probe changes in translational diffusivity of local water within 1 nm of nitroxide radical-based electron spin labels tethered to specific protein residues, and successfully reported on the study of protein folding, protein aggregation, and conformational changes of globular and membrane protein segments (18–21). ODNP has revealed increased heterogeneity, i.e., dispersion, in local water diffusivity on the surface of a folded protein, compared with its unfolded counterpart or folding intermediate (20). The basic connection between surface water diffusion and protein structural transformation is that conformational changes, locally or globally, are modulated by the shift in balance between protein surface−water versus protein−protein interactions. The varying stability of this local hydration shell will be collectively reflected in varying retardation of water diffusivity within about 1 nm of the protein surface of interest (22, 23). It has been reported that this surface water dynamics not only varies from surface to surface but is also dramatically heterogeneous from region to region on a given protein surface (20, 22). Terahertz measurements have shown that structural rearrangements of a protein at different folded states exhibit different Terahertz absorption energies that were attributed to changes in the coupling dynamics between the protein surface and hydration water (23). However, the heterogeneous water dynamics landscape of a structurally evolving or interacting protein in situ and in dilute solution has been elusive to probe to date. This study, aided by ODNP, sets out to exactly do that, namely, to probe the structural transition of tau in the early stages of aggregation in solution.Tau monomers belong to a family of intrinsically disordered proteins (IDPs) that, upon misfolding and aggregation, form highly structured amyloid fibrils (24). In this work, we studied the tau variant truncated between residues 255 and 441 (also known as Δtau187, Fig. 1A) of the longest isoform of the human tau protein (25) that includes all four microtubule-binding repeat domains (MTBD) and the C-terminal region (25, 26) (Fig. 1A). This and related truncated variants of tau have been shown to have greatly accelerated aggregation kinetics to form mature fibrils after approximately >12 h of aggregation (24–26). The known hydrophobic hexapeptide stretches, ²⁷⁵VQIINK²⁸⁰ (PHF6*) and ³⁰⁶VQIVYK³¹¹ (PHF6), found at the beginning of the second and third repeat segment are highly amyloidogenic, and form core regions of the tau fibrils (27, 28) with propensities for parallel in-register β-sheet structures reported (28–30). To induce tau fibrillization in vitro, polyanions, such as heparin or arachidonic acid micelles, are typically added to the solution (31). Although the molecular basis for this process is not fully understood, the structure of tau fibrils induced by heparin in vitro displays similar propensities to those found in AD patient tissues (31). Furthermore, heparin ultimately does not remain in the mature fibril core (32). Thus, heparin-induced tau aggregation has served as an accepted model for pathological tau fibrillization (30–32).Open in a separate windowFig. 1.Visualization of Δtau187 aggregates at different aggregation time (t). (A) Δtau187 consists of four microtubule-binding domains indicated as 1–4, where the amyloidogenic hexapeptide region of the third repeat is marked as a black box. The positions of the spin labels at mutated cysteine residues are indicated. (B) Cryo-TEM micrographs display the morphologies of Δtau187 (322C) aggregates after various aggregation times. (Scale bar, 100 nm.) (C) Cross-linked Δtau187 (322C) aggregates at different aggregation time analyzed by SDS/PAGE. (D) A schematic representation of the hypothesized Δtau187 species evolving at five aggregation stages. (E) Aggregation time dependence of τ of hydration water near different spin-labeled residues of Δtau187.In this study, we focused on capturing the structural transformation of the heparin-induced aggregation of Δtau187 in situ under ambient solution conditions and as a function of aggregation time. To achieve this, we used ODNP to probe the translational diffusion dynamics of surface water on protein surfaces and concurrently continuous wave (cw) electron spin resonance (ESR) line shape analysis (33, 34) to monitor protein side-chain mobility and interprotein contacts using the same protein sample (16, 17). Both methods rely on the site-directed mutagenesis and spin-labeling of the protein at a single-cysteine site with an (1-oxyl-2,2,5,5-tetramethylpyrrolidin-3-yl) methyl methanethiosulfonate (MTSL)-based nitroxide label, whose side chain is commonly termed “R1” (35). Δtau187 was singly R1 spin-labeled across several key residues of its third repeat MTBD and C terminus. The ODNP measurement was used to derive (as discussed in SI Overview of the ODNP Technique in detail) the translational correlation time, τ, of water within ∼1 nm of the R1 label tethered to the protein surface, together with two relaxivity parameters termed “k_σ” and “k_ρ” that report on contributions from freely diffusing water near the protein surface and bound water to the protein surface on the timescale of a few nanoseconds or longer, respectively. Thus, when a characteristic dispersion, i.e., an increased heterogeneity, of surface water diffusivity develops on the Δtau187 surface, as it transforms from monomeric species to oligomeric and larger fibrillar species, this indirectly shows that Δtau187 is undergoing structural transformation (18). However, ODNP analysis alone cannot differentiate between intraprotein or interprotein structural changes, nor does it offer population information. The cw ESR concurrently measures the structural and dynamic features of the same R1 spin-labeled protein species, as the backbone rearranges and interprotein contacts form during protein assembles. Specifically, we carried out quantitative ESR line shape simulation to determine the spin label mobility, and the population (percent) of the spin label in the respective motional states, and as embedded in parallel β-sheet arrangements. The spin label may experience distinct motional environments—slow vs. fast—that can be attributed to a local protein environment in which R1 is embedded at an intertau interface vs. one in which R1 is freely rotating in the solvent. Their evolving populations with increased aggregation time can be quantified. If R1 of Δtau187 at a specific position gets embedded in β-sheet structures, the evolving content of the β-sheets in which R1 labels of adjacent protein strands stack in parallel can be estimated by ESR line shape analysis. This is because, when packing in parallel β-sheet structures, the same R1-labeled sites from different protein strands come into intimate contact within 5–8 Å, yielding a characteristic single-line ESR spectral feature due to spin exchange between overlapping electron spin orbitals of the R1 labels (29, 36–41). We can verify the assignment of this spectral signature to parallel β-sheet stacking by systematic “spin dilution” to yield a mixture where only 20% of the tau strands are labeled with paramagnetic R1 labels and 80% are labeled with the diamagnetic analog of R1 labels, designated here as R1’, at a given site of Δtau187. This approach allows us to differentiate between the spin-exchanged and simply immobile spectral features, thereby confirming the origin of the characteristic spectral component as caused by β-sheet packing (36–41). Single-line, spin-exchanged, ESR spectra of protein fibrils have been previously assigned to parallel β-sheet structures (29, 36–41), including the mature fibrils of full-length tau proteins that were found to stack in parallel and in-register particularly around the PHF6 region (29).By combining ODNP and cw ESR measurements performed in situ and in solution together with conventional techniques of turbidimetry, Thioflavin T (ThT) staining fluorescence, transmission electron microscopy (TEM), and chemical cross-linking to probe aggregation of Δtau187, we aim to answer the following key questions: (i) At what stage of aggregation do structural transformations of tau proteins and their assembly occur? (ii) What are the structural and dynamic properties of the aggregation intermediates, and are they “on pathway” toward fibril formation? (iii) At what stage do stable β-sheet structures form, and what are their populations? (iv) Does fibril growth proceed mainly by elongation of preassembled oligomers or fibrils, or predominantly by monomer recruitment?     

Viscosity of deeply supercooled water and its coupling to molecular diffusion

The viscosity of a liquid measures its resistance to flow, with consequences for hydraulic machinery, locomotion of microorganisms, and flow of blood in vessels and sap in trees. Viscosity increases dramatically upon cooling, until dynamical arrest when a glassy state is reached. Water is a notoriously poor glassformer, and the supercooled liquid crystallizes easily, making the measurement of its viscosity a challenging task. Here we report viscosity of water supercooled close to the limit of homogeneous crystallization. Our values contradict earlier data. A single power law reproduces the 50-fold variation of viscosity up to the boiling point. Our results allow us to test the Stokes–Einstein and Stokes–Einstein–Debye relations that link viscosity, a macroscopic property, to the molecular translational and rotational diffusion, respectively. In molecular glassformers or liquid metals, the violation of the Stokes–Einstein relation signals the onset of spatially heterogeneous dynamics and collective motions. Although the viscosity of water strongly decouples from translational motion, a scaling with rotational motion remains, similar to canonical glassformers.Water, considered as a potential glassformer, has been a long-lasting topic of intense activity. Its possible liquid–glass transition was reported 50 years ago to be in the vicinity of 140?K (1, 2). However, ice nucleation hinders the access to this transition from the liquid side. Bypassing crystallization requires hyperquenching the liquid at tremendous cooling rates, ca. 10⁷?K ? s^?1 (3). As a consequence, many questions about supercooled and glassy water and its glass–liquid transition remain open (4–7).As an example, crystallization of water is accompanied by one of the largest known relative changes in sound velocity, which has been attributed to the relaxation effects of the hydrogen bond network (8, 9). Indeed, whereas the sound velocity is around 1,400 m⋅s−1 in liquid water at 273?K, it reaches around 3,300 m⋅s−1 in ice at 273?K and a similar value in the known amorphous phases of ice at 80?K (10). Such a large jump is usually the signature of a strong glass, i.e., one in which relaxation times or viscosity follow an Arrhenius law upon cooling. However, pioneering measurements on bulk supercooled water by NMR (11) and quasi-elastic neutron scattering (12), as well as recent ones by optical Kerr effect (8, 9), reveal a large super-Arrhenius behavior between 340 and 240?K, similar to what is observed in fragile glassformers (13, 14). The temperature dependence of the relaxation time is well described by a power law (8, 9), as expected from mode-coupling theory (15, 16), which usually applies well to liquids with a small change of sound velocity upon vitrification. Based on these and other observations, it has been hypothesized that supercooled water experiences a fragile-to-strong transition (17). This idea has motivated experimental efforts to measure dynamic properties of supercooled water and has received some indirect support from experiments on nanoconfined water (18–20) and from simulations (21, 22).In usual glassformers, many studies have focused on the coupling or decoupling between the following dynamic quantities: viscosity (η) and self or tracer diffusion coefficients for translation (D_t) and rotation (D_r). If objects as small as molecules were to follow macroscopic hydrodynamics, one would expect that the preceding quantities would be related through the Stokes–Einstein (SE), D_t ∝ T/η, and Stokes–Einstein–Debye (SED), D_r ∝ T/η, relations, where T is the temperature. These relations are indeed obeyed by many liquids at sufficiently high temperature. However, they might break down at low temperature. Pioneering experiments were performed by the groups of Sillescu (23–25) and Ediger (26–28) where a series of molecular glassformers were investigated. SE relation is obeyed at sufficiently high temperature but violated around 1.3T_g, where T_g is the glass transition temperature, thus indicating decoupling between translational diffusion and viscosity. In contrast, it was observed for ortho-terphenyl (23, 24, 26) that rotational diffusion and viscosity remain strongly coupled (i.e., obey the SED relation) even very close to T_g. A corollary is that translational and rotational diffusion decouple from each other at low temperature. These observations imply that deeply supercooled liquids exhibit spatially heterogeneous dynamics (29–31). Dynamic heterogeneities have been confirmed by direct observations of several single fluorescent molecules immersed in ortho-terphenyl (32) or nanorods immersed in glycerol (33). Physically different systems also show analogous behavior. Colloids near the colloidal glass transition violate SE but obey SED (34). In the metallic alloy Zr₆₄Ni₃₆, SE relation is even violated without supercooling, more than 35% above the liquidus temperature (35). This has also been related to the emergence of dynamic heterogeneities (36).For water, SE already breaks down at ambient temperature, which corresponds to around 2.1?T_g (T_g ? 136?K). Molecular dynamics simulations (37–39) have proposed that this occurs concurrently to dynamic heterogeneities caused by a putative liquid–liquid critical point. However, SE and SED also fail by application of high pressure at 400?K (40) where no liquid–liquid transition is expected. To gain more insight, the test of SE and SED in supercooled water deserves further investigation. Translational self-diffusion coefficient D_t (41) and rotational correlation time τ_r (assumed to scale as 1/D_r) (42) have thus been measured down to the homogeneous crystallization temperature (238?K) at ambient pressure. Their comparison reveals a decoupling between rotation and translation that increases with supercooling (42), similar to glassformers. However, viscosity data are needed for a direct test of SE and SED relations. Quite surprisingly, there are only two sets of data for the viscosity η at significant supercooling. Using Poiseuille flow in capillaries, Hallett (43) reached 249.35?K, and Osipov et al. (44) reached 238.15?K. However, the two sets disagree below 251?K, with an 8% difference at 249?K, beyond the reported uncertainties. The measurements in ref. 44 are suspected of errors (45) because of the small capillary diameter used. Here we report η at ambient pressure down to 239.27?K. Our study completes the knowledge of the main dynamic parameters of water down to the homogeneous crystallization limit and allows us to check the coupling of viscosity to molecular translation or rotation, as has been done for usual glassformers.     

Structural interactions of a voltage sensor toxin with lipid membranes

Protein toxins from tarantula venom alter the activity of diverse ion channel proteins, including voltage, stretch, and ligand-activated cation channels. Although tarantula toxins have been shown to partition into membranes, and the membrane is thought to play an important role in their activity, the structural interactions between these toxins and lipid membranes are poorly understood. Here, we use solid-state NMR and neutron diffraction to investigate the interactions between a voltage sensor toxin (VSTx1) and lipid membranes, with the goal of localizing the toxin in the membrane and determining its influence on membrane structure. Our results demonstrate that VSTx1 localizes to the headgroup region of lipid membranes and produces a thinning of the bilayer. The toxin orients such that many basic residues are in the aqueous phase, all three Trp residues adopt interfacial positions, and several hydrophobic residues are within the membrane interior. One remarkable feature of this preferred orientation is that the surface of the toxin that mediates binding to voltage sensors is ideally positioned within the lipid bilayer to favor complex formation between the toxin and the voltage sensor.Protein toxins from venomous organisms have been invaluable tools for studying the ion channel proteins they target. For example, in the case of voltage-activated potassium (Kv) channels, pore-blocking scorpion toxins were used to identify the pore-forming region of the channel (1, 2), and gating modifier tarantula toxins that bind to S1–S4 voltage-sensing domains have helped to identify structural motifs that move at the protein–lipid interface (3–5). In many instances, these toxin–channel interactions are highly specific, allowing them to be used in target validation and drug development (6–8).Tarantula toxins are a particularly interesting class of protein toxins that have been found to target all three families of voltage-activated cation channels (3, 9–12), stretch-activated cation channels (13–15), as well as ligand-gated ion channels as diverse as acid-sensing ion channels (ASIC) (16–21) and transient receptor potential (TRP) channels (22, 23). The tarantula toxins targeting these ion channels belong to the inhibitor cystine knot (ICK) family of venom toxins that are stabilized by three disulfide bonds at the core of the molecule (16, 17, 24–31). Although conventional tarantula toxins vary in length from 30 to 40 aa and contain one ICK motif, the recently discovered double-knot toxin (DkTx) that specifically targets TRPV1 channels contains two separable lobes, each containing its own ICK motif (22, 23).One unifying feature of all tarantula toxins studied thus far is that they act on ion channels by modifying the gating properties of the channel. The best studied of these are the tarantula toxins targeting voltage-activated cation channels, where the toxins bind to the S3b–S4 voltage sensor paddle motif (5, 32–36), a helix-turn-helix motif within S1–S4 voltage-sensing domains that moves in response to changes in membrane voltage (37–41). Toxins binding to S3b–S4 motifs can influence voltage sensor activation, opening and closing of the pore, or the process of inactivation (4, 5, 36, 42–46). The tarantula toxin PcTx1 can promote opening of ASIC channels at neutral pH (16, 18), and DkTx opens TRPV1 in the absence of other stimuli (22, 23), suggesting that these toxin stabilize open states of their target channels.For many of these tarantula toxins, the lipid membrane plays a key role in the mechanism of inhibition. Strong membrane partitioning has been demonstrated for a range of toxins targeting S1–S4 domains in voltage-activated channels (27, 44, 47–50), and for GsMTx4 (14, 50), a tarantula toxin that inhibits opening of stretch-activated cation channels in astrocytes, as well as the cloned stretch-activated Piezo1 channel (13, 15). In experiments on stretch-activated channels, both the d- and l-enantiomers of GsMTx4 are active (14, 50), implying that the toxin may not bind directly to the channel. In addition, both forms of the toxin alter the conductance and lifetimes of gramicidin channels (14), suggesting that the toxin inhibits stretch-activated channels by perturbing the interface between the membrane and the channel. In the case of Kv channels, the S1–S4 domains are embedded in the lipid bilayer and interact intimately with lipids (48, 51, 52) and modification in the lipid composition can dramatically alter gating of the channel (48, 53–56). In one study on the gating of the Kv2.1/Kv1.2 paddle chimera (53), the tarantula toxin VSTx1 was proposed to inhibit Kv channels by modifying the forces acting between the channel and the membrane. Although these studies implicate a key role for the membrane in the activity of Kv and stretch-activated channels, and for the action of tarantula toxins, the influence of the toxin on membrane structure and dynamics have not been directly examined. The goal of the present study was to localize a tarantula toxin in membranes using structural approaches and to investigate the influence of the toxin on the structure of the lipid bilayer.     

Highly permeable artificial water channels that can self-assemble into two-dimensional arrays

Bioinspired artificial water channels aim to combine the high permeability and selectivity of biological aquaporin (AQP) water channels with chemical stability. Here, we carefully characterized a class of artificial water channels, peptide-appended pillar[5]arenes (PAPs). The average single-channel osmotic water permeability for PAPs is 1.0(±0.3) × 10⁻¹⁴ cm³/s or 3.5(±1.0) × 10⁸ water molecules per s, which is in the range of AQPs (3.4∼40.3 × 10⁸ water molecules per s) and their current synthetic analogs, carbon nanotubes (CNTs, 9.0 × 10⁸ water molecules per s). This permeability is an order of magnitude higher than first-generation artificial water channels (20 to ∼10⁷ water molecules per s). Furthermore, within lipid bilayers, PAP channels can self-assemble into 2D arrays. Relevant to permeable membrane design, the pore density of PAP channel arrays (∼2.6 × 10⁵ pores per μm²) is two orders of magnitude higher than that of CNT membranes (0.1∼2.5 × 10³ pores per μm²). PAP channels thus combine the advantages of biological channels and CNTs and improve upon them through their relatively simple synthesis, chemical stability, and propensity to form arrays.The discovery of the high water and gas permeability of aquaporins (AQPs) and the development of artificial analogs, carbon nanotubes (CNTs), have led to an explosion in studies aimed at incorporating such channels into materials and devices for applications that use their unique transport properties (1–9). Areas of application include liquid and gas separations (10–13), drug delivery and screening (14), DNA recognition (15), and sensors (16). CNTs are promising channels because they conduct water and gas three to four orders of magnitude faster than predicted by conventional Hagen–Poiseuille flow theory (11). However, their use in large-scale applications has been hampered by difficulties in producing CNTs with subnanometer pore diameters and fabricating membranes in which the CNTs are vertically aligned (4). AQPs also efficiently conduct water across membranes (∼3 billion molecules per second) (17) and are therefore being studied intensively for their use in biomimetic membranes for water purification and other applications (1, 2, 18). The large-scale applications of AQPs is complicated by the high cost of membrane protein production, their low stability, and challenges in membrane fabrication (1).Artificial water channels, bioinspired analogs of AQPs created using synthetic chemistry (19), ideally have a structure that forms a water-permeable channel in the center and an outer surface that is compatible with a lipid membrane environment (1). Interest in artificial water channels has grown in recent years, following decades of research and focus on synthetic ion channels (19). However, two fundamental questions remain: (i) Can artificial channels approach the permeability and selectivity of AQP water channels? (ii) How can such artificial channels be packaged into materials with morphologies suitable for engineering applications?Because of the challenges in accurately replicating the functional elements of channel proteins, the water permeability and selectivity of first-generation artificial water channels were far below those of AQPs (SI Appendix, Table S1) (20–25). In some cases, the conduction rate for water was much lower than that of AQPs as a result of excess hydrogen bonds being formed between the water molecules and oxygen atoms lining the channel (20). The low water permeability that was measured for first-generation water channels also highlights the experimental challenge of accurately characterizing water flow through low-permeability water channels. Traditionally, a liposome-based technique has been used to measure water conduction, in which the response to an osmotic gradient is followed by measuring changes in light scattering (26, 27) or fluorescence (28). The measured rates are then converted to permeability values. These measurements suffer from a high background signal due to water diffusion through the lipid bilayer, which, in some cases, can be higher than water conduction through the inserted channels, making it challenging to resolve the permeability contributed by the channels (29). Thus, there is a critical need for a method to accurately measure single-channel permeability of artificial water channels to allow for accurate comparison with those of biological water channels. Furthermore, first-generation artificial water channels were designed with a focus on demonstrating water conduction and one-dimensional assembly into tubular structures (20–24), but how the channels could be assembled into materials suitable for use in engineering applications was not explored. To derive the most advantage from their fast and selective transport properties, artificial water channels are ideally vertically aligned and densely packed in a flat membrane. These features have been long desired but remain a challenge for CNT-based systems (4).Here we introduce peptide-appended pillar[5]arene (PAP; Fig. 1) (30) as an excellent architecture for artificial water channels, and we present data for their single-channel permeability and self-assembly properties. Nonpeptide pillar[5]arene derivatives were among first-generation artificial water channels (1, 23). Pillar[5]arene derivatives, including the one used in this study, have a pore of ∼5 Å in diameter and are excellent templates for functionalization into tubular structures (31–34). However, the permeability of hydrazide-appended pillar[5]arene channels was low (∼6 orders of magnitude lower than that of AQPs; SI Appendix, Table S1). We addressed the challenges of accurately measuring single-channel water permeability and improving the water conduction rate over first-generation artificial water channels by using both experimental and simulation approaches. The presented PAP channel contains more hydrophobic regions (30) compared with its predecessor channel (23), which improves both its water permeability and its ability to insert into membranes. To determine single-channel permeability of PAPs, we combined stopped-flow light-scattering measurements of lipid vesicles containing PAPs with fluorescence correlation spectroscopy (FCS) (35, 36). Stopped-flow experiments allow the kinetics of vesicle swelling or shrinking to be followed with millisecond resolution and water permeability to be calculated, whereas FCS makes it possible to count the number of channels per vesicle (36, 37). The combination of the two techniques allows molecular characterization of channel properties with high resolution and demonstrates that PAP channels have a water permeability close to those of AQPs and CNTs. The experimental results were corroborated by molecular dynamics (MD) simulations, which also provided additional insights into orientation and aggregation of the channels in lipid membranes. Finally, as a first step toward engineering applications such as liquid and gas separations, we were able to assemble PAP channels into highly packed planar membranes, and we experimentally confirmed that the channels form 2D arrays in these membranes.Open in a separate windowFig. 1.Structure of the peptide-appended pillar[5]arene (PAP) channel. (A) The PAP channel (C₃₂₅H₃₂₀N₃₀O₆₀) forms a pentameric tubular structure through intramolecular hydrogen bonding between adjacent alternating d-l-d phenylalanine chains (d-Phe-l-Phe-d-Phe-COOH). (B) Molecular modeling (Gaussian09, semiempirical, PM6) of the PAP channel shows that the benzyl rings of the phenylalanine side chains extend outward from the channel walls (C, purple; H, white; O, red; N, blue). (C and D) MD simulation of the PAP channel in a POPC bilayer revealed its interactions with the surrounding lipids. The five chain-like units of the channel are colored purple, blue, ochre, green, and violet, with hydrogen atoms omitted. In C, the POPC lipids are represented by thin tan lines; in D, water is shown as red (oxygen) and white (hydrogen) van der Waals spheres.     

INAUGURAL ARTICLE by a Recently Elected Academy Member:Photogeneration of hydrogen from water using CdSe nanocrystals demonstrating the importance of surface exchange

Unique tripodal S-donor capping agents with an attached carboxylate are found to bind tightly to the surface of CdSe nanocrystals (NCs), making the latter water soluble. Unlike that in similarly solubilized CdSe NCs with one-sulfur or two-sulfur capping agents, dissociation from the NC surface is greatly reduced. The impact of this behavior is seen in the photochemical generation of H₂ in which the CdSe NCs function as the light absorber with metal complexes in aqueous solution as the H₂-forming catalyst and ascorbic acid as the electron donor source. This precious-metal–free system for H₂ generation from water using [Co(bdt)₂]⁻ (bdt, benzene-1,2-dithiolate) as the catalyst exhibits excellent activity with a quantum yield for H₂ formation of 24% at 520 nm light and durability with >300,000 turnovers relative to catalyst in 60 h.Artificial photosynthesis (AP) represents an important strategy for energy conversion from sunlight to storage in chemical bonds (1–4). Unlike natural photosynthesis in which CO₂ + H₂O are converted into carbohydrates and O₂, the key energy-storing reaction in AP is the splitting of water into its constituent elements of hydrogen and oxygen (5–16). As a redox reaction, water splitting can be divided into two half-reactions, of which the light-driven generation of H₂ is the reductive component. Many systems for the photogeneration of H₂ have been described over the years and they typically consist of a light absorber, a catalyst for H₂ formation, and sources of protons and electrons. For systems that function in aqueous media, the protons are provided by water, whereas for nonaqueous systems, the protons are provided by weak, generally organic acids. The source of electrons in these photochemical systems is generally a sacrificial electron donor—that is, a compound that decomposes following one electron oxidation.Reports of the light-driven generation of hydrogen date back more than 30 y, beginning with a multicomponent system containing [Ru(bpy)₃]²⁺ (where bpy is 2,2′-bipyridine) as the chromophore or photosensitizer (PS) and colloidal Pt as the catalyst for making H₂ from protons and electrons (17). In these and many subsequent systems, electron mediators were used to accept an electron from the excited chromophore, PS*— thereby serving as an oxidative quencher—and transfer it to the catalyst. Whereas two of the initial mediators were bpy complexes of rhodium and cobalt (17, 18), the overwhelming majority of electron mediators in these systems were dialkylated 2,2′- and 4,4′-bipyridines and their derivatives (19–22). The most extensively used of these mediators was methyl viologen (MV²⁺, dimethyl-4,4′-bipyridinium, usually as its chloride salt). These mediators were subsequently found to undergo deactivation in their role by hydrogenation (23, 24). The sacrificial electron donors used in these studies depended on system pH and were generally based on compounds having tertiary amine functionality for decomposition following oxidation, such as triethylamine (TEA), triethanolamine (TEOA), and ethylenediamine-N,N,N′,N′-tetraacetic acid (EDTA) (17, 19–22). A different electron mediator during the early studies on light-driven generation of hydrogen was found to be TiO₂, which when platinized served as both the mediator and the catalyst (25–28).During the more than three decades that have passed since the initial reports (17, 19–22, 25–27), every aspect and component of photochemical proton reduction systems have been investigated with the goal of increasing activity and durability. These include new molecular catalysts and different photosensitizers ranging from other metal complexes with long-lived charge-transfer excited states to strongly absorbing organic dyes. With a view toward the possible long-term utilization of hydrogen from solar-driven water splitting, efforts have expanded over the past decade to use components that contain only earth-abundant elements and thus to remove Pt, Pd, Ru, Ir, and Rh from such systems. In this regard, photochemical proton reduction systems have been reported in which complexes of cobalt, nickel, and iron are found to function as catalysts for hydrogen generation (18, 29–41). A number of these complexes were inspired by the active sites of hydrogenase enzymes in which Fe is, and Ni may be, present, and a pendant organic base is thought to help as a proton shuttle to a postulated metal-hydride intermediate for H₂ formation (30, 31, 35, 37, 42, 43).Another set of complexes investigated as catalysts for proton reduction are complexes of Co having diglyoxime-type ligands that form a pseudomacrocyclic structure (that is, two diglyoxime ligands linked together by either H bonds or BF₂ bridges) (44–54). Although many of these studies with regard to catalyst development were, and are, based on electrocatalytic generation of H₂ (44–50), more recent efforts have used the cobaloxime catalysts in light-driven systems (51–54). Photosensitizers in these investigations have been either charge transfer metal complexes of Ru(II), Ir(III), Re(I), and Pt(II) or organic dyes. Although some of these systems exhibited significant activity for making H₂, all of them suffered from instability that led to cessation of activity after periods ranging from 6 h to 30 h.The use of the cobaloxime catalyst CoCl(pyr)(dmg)₂ (where dmg is dimethylglyoximate anion) in conjunction with organic dyes as PS provided the first molecular systems for visible light-driven proton reduction to H₂ that were free of precious metals (55–57). The most effective of these used a Se-derivatized rhodamine dye as the chromophore with TEOA as the sacrificial electron donor, yielding good activity with an initial turnover frequency (TOF) > 5,000/h (vs. PS) and total turnover number (TON) of 9,000 after 8 h (57). Analysis of this system revealed that it functioned via reductive quenching of PS* by TEOA and subsequent electron transfer from PS^– to the catalyst. Another organic dye-catalyst system that also exhibited good activity used fluorescein (Fl) as PS and a nickel pyridinethiolate (pyS) catalyst in pH 11 media with TEA as the sacrificial donor. This system also was found to function via reductive quenching of PS* by the electron donor, rather than by direct electron transfer from PS* to the catalyst or an electron mediator (37). A significant number of reviews provide detailed accounts of the various systems studied and their effectiveness with regard to H₂ generation (1, 58–64). However, all of them, which contain molecular light absorbers [charge transfer (CT) metal complexes and organic dyes], suffer from photoinstability during prolonged irradiation. Additionally, the molecular catalysts for H₂ generation may undergo deactivation, as has been established for the Co glyoximate complexes.In an analysis for genuinely viable systems for proton reduction and water oxidation in solar-driven water splitting, Bard and Fox addressed the question of component stability and indicated a need to focus on the use of semiconductors (SCs) as light absorbers based on the wide energy range of SC bandgaps, the electron transfer properties of excited semiconductors, and their potential stability under prolonged irradiation (65). Although the use of semiconductors for photochemical water splitting dates back to a report by Fujishima and Honda in 1972 with TiO₂ and UV light, the challenge was to use SCs with absorption maxima that better matched the solar spectrum (66). There have been numerous reports describing efforts in this direction and several recent reviews offer a summary of systems used and results obtained (67–75). Semiconductor nanoparticles that exhibit size-constrained electronic properties represent a large and important class of possible light absorbers for the two half-reactions of water splitting. These nanoparticles, which are referred to as quantum dots (QDs) and nanocrystals (NCs), represent a fertile area of study in the context of energy conversion because their bandgaps can be adjusted via their preparation and their solubility can be controlled by their surface stabilizers or capping agents (76). In this way, NCs can offer unique size-dependent optical properties and stronger light absorption over a wider spectral range than do molecular PSs (68, 76). In fact, NCs as light absorbers in combination with precious metal proton reduction catalysts or with Fe-Fe hydrogenase have been studied, yielding interesting photocatalytic systems (77–81).We recently communicated such a system for carrying out H₂ formation from aqueous protons that possessed great durability and impressive activity. The light absorber in this system was water-solubilized CdSe NCs, the catalyst was an in situ-formed complex of Ni²⁺ with the water-solubilizing agent dihydrolipoic acid (DHLA), the electron source was ascorbic acid (AA), and the system medium was water at pH 4.5. TONs of more than 600,000 were reported for one set of conditions, using 520 nm light, whereas for a different set of conditions durability over 15 d was found (82). Water solubilization of CdSe NCs using agents such as 3-thiopropionic acid and DHLA has been known for some time, with DHLA more strongly binding via chelation (83–85). In the system that we previously reported for H₂ production, however, dissociation of DHLA from the CdSe NCs was an essential aspect of its operation to form the Ni-DHLA catalyst (82). On the other hand, the dissociation of DHLA from the CdSe NCs was also found to negatively affect the examination of preformed catalysts because of competing exchange reactions involving DHLA and the catalyst ligands.In our current study, we report a unique hydrogen-generating system using CdSe NCs with much less labile water-solubilizing capping agents. This unique system, which is more durable, allows assessment of the activity of successful H₂-generating catalysts that had been established electrochemically or in a different photochemical system. The reduced lability of the water-solubilizing agent is based on having three S donors in close proximity to each other for the formation of a more stable bridging structure to the CdSe nanoparticle.     

From the Cover: The point of no return in vetoing self-initiated movements

In humans, spontaneous movements are often preceded by early brain signals. One such signal is the readiness potential (RP) that gradually arises within the last second preceding a movement. An important question is whether people are able to cancel movements after the elicitation of such RPs, and if so until which point in time. Here, subjects played a game where they tried to press a button to earn points in a challenge with a brain–computer interface (BCI) that had been trained to detect their RPs in real time and to emit stop signals. Our data suggest that subjects can still veto a movement even after the onset of the RP. Cancellation of movements was possible if stop signals occurred earlier than 200 ms before movement onset, thus constituting a point of no return.It has been repeatedly shown that spontaneous movements are preceded by early brain signals (1–8). As early as a second before a simple voluntary movement, a so-called readiness potential (RP) is observed over motor-related brain regions (1–3, 5). The RP was found to precede the self-reported time of the “‘decision’ to act” (ref. 3, p. 623). Similar preparatory signals have been observed using invasive electrophysiology (8, 9) and functional MRI (7, 10), and have been demonstrated also for choices between multiple-response options (6, 7, 10), for abstract decisions (10), for perceptual choices (11), and for value-based decisions (12). To date, the exact nature and causal role of such early signals in decision making is debated (12–20).One important question is whether a person can still exert a veto by inhibiting the movement after onset of the RP (13, 18, 21, 22). One possibility is that the onset of the RP triggers a causal chain of events that unfolds in time and cannot be cancelled. The onset of the RP in this case would be akin to tipping the first stone in a row of dominoes. If there is no chance of intervening, the dominoes will gradually fall one-by-one until the last one is reached. This has been coined a ballistic stage of processing (23, 24). A different possibility is that participants can still terminate the process, akin to taking out a domino at some later stage in the chain and thus preventing the process from completing. Here, we directly tested this in a real-time experiment that required subjects to terminate their decision to move once a RP had been detected by a brain–computer interface (BCI) (25–31).     

Kinetics of nucleotide-dependent structural transitions in the kinesin-1 hydrolysis cycle

To dissect the kinetics of structural transitions underlying the stepping cycle of kinesin-1 at physiological ATP, we used interferometric scattering microscopy to track the position of gold nanoparticles attached to individual motor domains in processively stepping dimers. Labeled heads resided stably at positions 16.4 nm apart, corresponding to a microtubule-bound state, and at a previously unseen intermediate position, corresponding to a tethered state. The chemical transitions underlying these structural transitions were identified by varying nucleotide conditions and carrying out parallel stopped-flow kinetics assays. At saturating ATP, kinesin-1 spends half of each stepping cycle with one head bound, specifying a structural state for each of two rate-limiting transitions. Analysis of stepping kinetics in varying nucleotides shows that ATP binding is required to properly enter the one-head–bound state, and hydrolysis is necessary to exit it at a physiological rate. These transitions differ from the standard model in which ATP binding drives full docking of the flexible neck linker domain of the motor. Thus, this work defines a consensus sequence of mechanochemical transitions that can be used to understand functional diversity across the kinesin superfamily.Kinesin-1 is a motor protein that steps processively toward microtubule plus-ends, tracking single protofilaments and hydrolyzing one ATP molecule per step (1–6). Step sizes corresponding to the tubulin dimer spacing of 8.2 nm are observed when the molecule is labeled by its C-terminal tail (7–10) and to a two-dimer spacing of 16.4 nm when a single motor domain is labeled (4, 11, 12), consistent with the motor walking in a hand-over-hand fashion. Kinesin has served as an important model system for advancing single-molecule techniques (7–10) and is clinically relevant for its role in neurodegenerative diseases (13), making dissection of its step a popular ongoing target of study.Despite decades of work, many essential components of the mechanochemical cycle remain disputed, including (i) how much time kinesin-1 spends in a one-head–bound (1HB) state when stepping at physiological ATP concentrations, (ii) whether the motor waits for ATP in a 1HB or two-heads–bound (2HB) state, and (iii) whether ATP hydrolysis occurs before or after tethered head attachment (4, 11, 14–20). These questions are important because they are fundamental to the mechanism by which kinesins harness nucleotide-dependent structural changes to generate mechanical force in a manner optimized for their specific cellular tasks. Addressing these questions requires characterizing a transient 1HB state in the stepping cycle in which the unattached head is located between successive binding sites on the microtubule. This 1HB intermediate is associated with the force-generating powerstroke of the motor and underlies the detachment pathway that limits motor processivity. Optical trapping (7, 19, 21, 22) and single-molecule tracking studies (4, 8–11) have failed to detect this 1HB state during stepping. Single-molecule fluorescence approaches have detected a 1HB intermediate at limiting ATP concentrations (11, 12, 14, 15), but apart from one study that used autocorrelation analysis to detect a 3-ms intermediate (17), the 1HB state has been undetectable at physiological ATP concentrations.Single-molecule microscopy is a powerful tool for studying the kinetics of structural changes in macromolecules (23). Tracking steps and potential substeps for kinesin-1 at saturating ATP has until now been hampered by the high stepping rates of the motor (up to 100 s⁻¹), which necessitates high frame rates, and the small step size (8.2 nm), which necessitates high spatial precision (7). Here, we apply interferometric scattering microscopy (iSCAT), a recently established single-molecule tool with high spatiotemporal resolution (24–27) to directly visualize the structural changes underlying kinesin stepping. By labeling one motor domain in a dimeric motor, we detect a 1HB intermediate state in which the tethered head resides over the bound head for half the duration of the stepping cycle at saturating ATP. We further show that at physiological stepping rates, ATP binding is required to enter this 1HB state and that ATP hydrolysis is required to exit it. This work leads to a significant revision of the sequence and kinetics of mechanochemical transitions that make up the kinesin-1 stepping cycle and provides a framework for understanding functional diversity across the kinesin superfamily.     

Mobile hydrogen carbonate acts as proton acceptor in photosynthetic water oxidation

Cyanobacteria, algae, and plants oxidize water to the O₂ we breathe, and consume CO₂ during the synthesis of biomass. Although these vital processes are functionally and structurally well separated in photosynthetic organisms, there is a long-debated role for CO₂/ in water oxidation. Using membrane-inlet mass spectrometry we demonstrate that acts as a mobile proton acceptor that helps to transport the protons produced inside of photosystem II by water oxidation out into the chloroplast’s lumen, resulting in a light-driven production of O₂ and CO₂. Depletion of from the media leads, in the absence of added buffers, to a reversible down-regulation of O₂ production by about 20%. These findings add a previously unidentified component to the regulatory network of oxygenic photosynthesis and conclude the more than 50-y-long quest for the function of CO₂/ in photosynthetic water oxidation.Oxygenic photosynthesis in cyanobacteria, algae, and higher plants leads to the reduction of atmospheric CO₂ to energy-rich carbohydrates. The electrons needed for this process are extracted in a cyclic, light-driven process from water that is split into dioxygen (O₂) and protons. This reaction is catalyzed by a penta-µ-oxo bridged tetra-manganese calcium cluster (Mn₄CaO₅) within the oxygen-evolving complex (OEC) of photosystem II (PSII) (1–4). The possible roles of inorganic carbon, , in this process have been a controversial issue ever since Otto Warburg and Günter Krippahl (5) reported in 1958 that oxygen evolution by PSII strictly depends on CO₂ and therefore has to be based on the photolysis of H₂CO₃ (“Kohlensäure”) and not of water. These first experiments were indirect and, as became apparent later, were wrongly interpreted (6–8). Several research groups followed up on these initial results and identified two possible sites of C_i interaction within PSII (reviewed in refs. 9–12). Functional and spectroscopic studies showed that facilitates the reduction of the secondary plastoquinone electron acceptor (Q_B) of PSII by participating in the protonation of . Binding of (or ) to the nonheme Fe between the quinones Q_A and Q_B was recently confirmed by X-ray crystallography (3, 13, 14). Despite this functional role at the acceptor side, the very tight binding of to this site makes it impossible for the activity of PSII to be affected by changing the C_i level of the medium; instead inhibitors such as formate need to be added to induce the acceptor-side effect (15). Consequently, the water-splitting electron-donor side of PSII has also been studied intensively (for recent reviews, see refs. 11 and 12). Although a tight binding of C_i near the Mn₄CaO₅ cluster is excluded on the basis of X-ray crystallography (3, 14), FTIR spectroscopy (16), and mass spectrometry (17, 18), the possibility that a weakly bound affects the activity of PSII at the donor side remains a viable option (reviewed in refs. 10 and 19).In the present study using higher plant PSII membranes, we specifically evaluate a recently suggested role of weakly bound , namely, that it acts as an acceptor for, and transporter of, protons produced by water splitting in the OEC (20–22).     

Microscopic identification of the order parameter governing liquid–liquid transition in a molecular liquid

A liquid–liquid transition (LLT) in a single-component substance is an unconventional phase transition from one liquid to another. LLT has recently attracted considerable attention because of its fundamental importance in our understanding of the liquid state. To access the order parameter governing LLT from a microscopic viewpoint, here we follow the structural evolution during the LLT of an organic molecular liquid, triphenyl phosphite (TPP), by time-resolved small- and wide-angle X-ray scattering measurements. We find that locally favored clusters, whose characteristic size is a few nanometers, are spontaneously formed and their number density monotonically increases during LLT. This strongly suggests that the order parameter of LLT is the number density of locally favored structures and of nonconserved nature. We also show that the locally favored structures are distinct from the crystal structure and these two types of orderings compete with each other. Thus, our study not only experimentally identifies the structural order parameter governing LLT, but also may settle a long-standing debate on the nature of the transition in TPP, i.e., whether the transition is LLT or merely microcrystal formation.Liquid-liquid transition (LLT) is an intriguing phenomenon in which a liquid transforms into another one via a first-order transition. This means that there can be more than two liquid states for a single-component substance. Despite its counterintuitive nature, there have recently been many pieces of experimental and numerical evidence for the existence of LLT, for various liquids such as water (1–5), aqueous solutions (6–8), triphenyl phosphite (9–12), l-butanol (13), phosphorus (14), silicon (15, 16), germanium (17), and Y₂O₃–Al₂O₃ (18, 19). This suggests that the LLT may be rather universally observed for various types of liquids. However, none of the LLTs reported so far is free from criticisms (20, 21), mainly because these LLTs take place under experimentally difficult conditions [e.g., at high temperature and pressure (14, 15, 17–19)] or in a supercooled state below the melting point (1–3, 5–7, 9, 10), where the transition is inevitably contaminated by microcrystal formation. The latter is not limited to experiments but arises in numerical simulations, often causing many controversies [LLT (22–25) vs. crystallization (26–28)]. For ST2 water, however, this issue has recently been settled by an extensive simulation study by Palmer et al. (4).One of the hottest and long-standing debates is on the nature of the transition found in a molecular liquid, triphenyl phosphite (TPP), by Kivelson and his coworkers (29). The transition is very easy to access experimentally, because it takes place at ambient pressure and at a temperature range between 230 and 210 K and the transformation speed is slow enough to follow the kinetics. Since the finding of this transition (29, 30), many researchers thus have been interested in this intriguing phenomenon and there have been hot discussions on the nature of the transition (20, 21). Some people interpreted this as a liquid-associated phenomenon (9, 10, 31, 32), but others interpret it differently. All of the controversies come from the fact that this transition accompanies microcrystal formation and thus the final state, which is called “glacial phase,” often contains microcrystallites. This led many researchers to explain the transition by non-LLT scenarios, which include a defect-ordered phase scenario predicted by a frustration limited domain theory (29, 30, 33, 34), a microcrystallization scenario (35–38), and a liquid-crystal or plastic-crystal phase scenario (39). Each scenario captures a certain feature of the glacial phase, but fails in explaining all of the experimental results in a consistent manner. Similar situations are often seen in other candidates of LLTs, such as l-butanol [LLT (13) vs. microcrystallization (40–43)], confined water [LLT (5) vs. other phenomena (44–46)], and aqueous solutions [LLT (6, 7) vs. microcrystallization (8, 28, 47, 48)]. For TPP, however, some pieces of experimental evidence supportive of the LLT scenario rather than the microcrystallization scenario have recently been reported (11, 12).We propose a two-order-parameter (TOP) model of a liquid to explain LLT (20, 49). The main point of this model is that it is necessary to consider the spatiotemporal hierarchical nature of a liquid to understand LLT. More specifically, we argue that in addition to density order parameter ρ describing a gas–liquid transition, we need an additional scalar order parameter S, which is the number density of locally favored structures (LFS). In this model, LLT is a consequence of the cooperative ordering of the scalar nonconserved order parameter S, i.e., the cooperative formation of LFS. In other words, LLT is regarded as a gas–liquid-like transition of LFS: one liquid is a gas state of LFS (low-S state), and the other is its liquid state (high-S state). Recently, it was proposed by Anisimov and coworkers (50, 51) that the thermodynamic ordering field conjugate to the order parameter is the conversion equilibrium constant, which further characterizes the nature of LLT. We explained our experimental observation of LLT in TPP in terms of this model (9, 10). We also studied the phase transition dynamics and the physical and chemical properties of the second liquid state (liquid II), which were also explained by the model (20, 21).However, we have not had any direct experimental evidence for the formation of such LFS up to now; thus, an open question is, what is the relevant order parameter governing LLT, although the link of the order parameter to the enthalpy (9, 10), the refractive index (or, density) (9, 10, 29, 30), and the polarity associated with local molecular ordering (12) has been suggested for LLT in TPP. There have been structural studies on LLT by X-ray and neutron scattering measurements, focusing on local liquid structures at an inter- and intramolecular scale (36, 38, 52–54) and mesoscopic structures (34, 55). However, there has been no experimental evidence for the presence of locally favored structures, which characterize the liquid state uniquely, or the order parameter has still not been identified from a microscopic viewpoint.Here we study the structural change of TPP during LLT by time-resolved small- and wide-angle X-ray scattering measurements, which cover a length scale from a single molecule size ( ～  1 nm) to more than tens of nanometers. We show, to our knowledge, the first direct evidence for the presence of LFS and the temporal increase upon the liquid I-to-liquid II transformation. Furthermore, we also find an indication of the formation of microcrystallites during LLT. However, we reveal that LFS and microcrystallites have different sizes and growth kinetics, indicating that although they sometimes appear simultaneously during the process of LLT, LLT itself is driven by the formation of LFS and not by that of microcrystallites. We also discover that LFS are destroyed upon crystallization, clearly indicating not only that these two types of orderings are competing with each other but also that LFS is a structure unique to the liquid state. Our findings provide a comprehensive view on the long-standing controversy on the origin of the glacial phase, which was discovered by Kivelson and his coworkers (29, 30), and show that the fraction of LFS may be the relevant order parameter of LLT. This suggests that a liquid can have a spatiotemporal hierarchical structure at a low temperature, contrary to the common picture of a high-temperature liquid where the structure is random and homogeneous beyond the molecular size.     

α-Synuclein assembles into higher-order multimers upon membrane binding to promote SNARE complex formation

Physiologically, α-synuclein chaperones soluble NSF attachment protein receptor (SNARE) complex assembly and may also perform other functions; pathologically, in contrast, α-synuclein misfolds into neurotoxic aggregates that mediate neurodegeneration and propagate between neurons. In neurons, α-synuclein exists in an equilibrium between cytosolic and membrane-bound states. Cytosolic α-synuclein appears to be natively unfolded, whereas membrane-bound α-synuclein adopts an α-helical conformation. Although the majority of studies showed that cytosolic α-synuclein is monomeric, it is unknown whether membrane-bound α-synuclein is also monomeric, and whether chaperoning of SNARE complex assembly by α-synuclein involves its cytosolic or membrane-bound state. Here, we show using chemical cross-linking and fluorescence resonance energy transfer (FRET) that α-synuclein multimerizes into large homomeric complexes upon membrane binding. The FRET experiments indicated that the multimers of membrane-bound α-synuclein exhibit defined intermolecular contacts, suggesting an ordered array. Moreover, we demonstrate that α-synuclein promotes SNARE complex assembly at the presynaptic plasma membrane in its multimeric membrane-bound state, but not in its monomeric cytosolic state. Our data delineate a folding pathway for α-synuclein that ranges from a monomeric, natively unfolded form in cytosol to a physiologically functional, multimeric form upon membrane binding, and show that only the latter but not the former acts as a SNARE complex chaperone at the presynaptic terminal, and may protect against neurodegeneration.α-Synuclein is an abundant presynaptic protein that physiologically acts to promote soluble NSF attachment protein receptor (SNARE) complex assembly in vitro and in vivo (1–3). Point mutations in α-synuclein (A30P, E46K, H50Q, G51D, and A53T) as well as α-synuclein gene duplications and triplications produce early-onset Parkinson''s disease (PD) (4–10). Moreover, α-synuclein is a major component of intracellular protein aggregates called Lewy bodies, which are pathological hallmarks of neurodegenerative disorders such as PD, Lewy body dementia, and multiple system atrophy (11–14). Strikingly, neurotoxic α-synuclein aggregates propagate between neurons during neurodegeneration, suggesting that such α-synuclein aggregates are not only intrinsically neurotoxic but also nucleate additional fibrillization (15–18).α-Synuclein is highly concentrated in presynaptic terminals where α-synuclein exists in an equilibrium between a soluble and a membrane-bound state, and is associated with synaptic vesicles (19–22). The labile association of α-synuclein with membranes (23, 24) suggests that binding of α-synuclein to synaptic vesicles, and its dissociation from these vesicles, may regulate its physiological function. Membrane-bound α-synuclein assumes an α-helical conformation (25–32), whereas cytosolic α-synuclein is natively unfolded and monomeric (refs. 25, 26, 31, and 32; however, see refs. 33 and 34 and Discussion for a divergent view). Membrane binding by α-synuclein is likely physiologically important because in in vitro experiments, α-synuclein remodels membranes (35, 36), influences lipid packing (37, 38), and induces vesicle clustering (39). Moreover, membranes were found to be important for the neuropathological effects of α-synuclein (40–44).However, the relation of membrane binding to the in vivo function of α-synuclein remains unexplored, and it is unknown whether α-synuclein binds to membranes as a monomer or oligomer. Thus, in the present study we have investigated the nature of the membrane-bound state of α-synuclein and its relation to its physiological function in SNARE complex assembly. We found that soluble monomeric α-synuclein assembles into higher-order multimers upon membrane binding and that membrane binding of α-synuclein is required for its physiological activity in promoting SNARE complex assembly at the synapse.     

Anomalous water diffusion in salt solutions

The dynamics of water exhibits anomalous behavior in the presence of different electrolytes. Recent experiments [Kim JS, Wu Z, Morrow AR, Yethiraj A, Yethiraj A (2012) J Phys Chem B 116(39):12007–12013] have found that the self-diffusion of water can either be enhanced or suppressed around CsI and NaCl, respectively, relative to that of neat water. Here we show that unlike classical empirical potentials, ab initio molecular dynamics simulations successfully reproduce the qualitative trends observed experimentally. These types of phenomena have often been rationalized in terms of the “structure-making” or “structure-breaking” effects of different ions on the solvent, although the microscopic origins of these features have remained elusive. Rather than disrupting the network in a significant manner, the electrolytes studied here cause rather subtle changes in both structural and dynamical properties of water. In particular, we show that water in the ab initio molecular dynamics simulations is characterized by dynamic heterogeneity, which turns out to be critical in reproducing the experimental trends.Despite being one of the most-studied liquids, the properties of water and the nature of its interactions with other physical systems continue to be at the forefront of current research in many fields of science (1–8). One important facet of this vast research is the role of water in the solvation of ions. Thus, understanding the effect that ions have on the structural and dynamical properties of water has been a subject of numerous experimental and theoretical studies (6, 9–23). Besides being a classical textbook problem in physical chemistry, the coupling between solutes such as ions and molecules and the surrounding solvent has deep implications on a plethora of biologically relevant processes (24–26).Over six decades ago, Gurney introduced the notion of “structure makers” and “structure breakers” within the context of how different ions would perturb water’s hydrogen bond (HB) network (27). These ideas have generally been accepted and applied to explain various phenomena observed in electrolyte solutions (6, 28). One such example is the celebrated Hofmeister series, a list of cations and anions empirically discovered by Hofmeister, who found that different ions have varying tendencies to salt-out proteins from solution (6, 26). Although there exist some similarities between this series and various phenomenological measures of structure making and breaking, how exactly the structure of water and the extent of hydrogen bonding should be measured remains an open problem. Experimental studies from the Bakker group (13, 23) probing the rotational mobility of water, for example, have in fact suggested that the presence of ions does not even result in the enhancement or breakdown of the HB network of liquid water.Perhaps more interesting are questions concerning the connection between structural perturbations and the changes that ions induce on the dynamical properties of water. One important measure of this effect that will form the focus of this study is the self-diffusion coefficient of water molecules (D_W) in electrolyte solutions. In particular, NMR experiments have shown that below 3 M salt concentration, D_W for electrolytes like CsI increases as a function of concentration whereas the opposite trend is observed for NaCl (29). Our interest in these experiments is also piqued by the fact that recent molecular dynamics (MD) simulations using both fixed charge and polarizable force fields (FF) of the same systems do not succeed in even qualitatively predicting the experimental trends––D_W decreased with salt concentration for all of the systems studied (29)! We cannot exclude the possibility that an empirical potential can be constructed to reproduce these phenomena. However, our results from this work raise serious concerns about the use of empirical potentials in simulating electrolyte solutions in different applications and hence fail to provide a model that could be used to get a better understanding of the microscopic origins behind the anomalous water diffusion.Herein we revisit this problem using state-of-the-art ab initio molecular dynamics (AIMD) simulations where the electronic degrees of freedom are explicitly treated. Unlike the empirical simulations, we find that the AIMD qualitatively reproduce the trends observed in the experimental D_W. First, our analysis of various dynamical properties, such as residence times, unequivocally shows that there is a characteristic dynamic heterogeneity in the water ensemble that is present in the AIMD but absent in the empirical simulations. Rather than inducing significant perturbations to the dynamical properties, we find that the ions result in subtle but measurable changes in the tails of the dynamical ensemble. Although our analysis of various structural features indicates that there are effects that could be likened to structure making and breaking, the HB network is not disrupted or broken in any significant manner. In similar spirit to some recent work from our group that looked at directional correlations in the HB network relevant for proton and hydroxide diffusion (7), we show that ions such as Na⁺, Cl⁻, Cs⁺, and I⁻ substitute the role of water molecules in the network participating in directed ring structures with similar “network rules” present in neat water. The AIMD and empirical HB network exhibit qualitative differences which provide clues into the origins of the discrepancies previously noted.     

Therapeutic mechanisms of high-frequency stimulation in Parkinson’s disease and neural restoration via loop-based reinforcement

High-frequency deep brain stimulation (HFS) is clinically recognized to treat parkinsonian movement disorders, but its mechanisms remain elusive. Current hypotheses suggest that the therapeutic merit of HFS stems from increasing the regularity of the firing patterns in the basal ganglia (BG). Although this is consistent with experiments in humans and animal models of Parkinsonism, it is unclear how the pattern regularization would originate from HFS. To address this question, we built a computational model of the cortico-BG-thalamo-cortical loop in normal and parkinsonian conditions. We simulated the effects of subthalamic deep brain stimulation both proximally to the stimulation site and distally through orthodromic and antidromic mechanisms for several stimulation frequencies (20–180 Hz) and, correspondingly, we studied the evolution of the firing patterns in the loop. The model closely reproduced experimental evidence for each structure in the loop and showed that neither the proximal effects nor the distal effects individually account for the observed pattern changes, whereas the combined impact of these effects increases with the stimulation frequency and becomes significant for HFS. Perturbations evoked proximally and distally propagate along the loop, rendezvous in the striatum, and, for HFS, positively overlap (reinforcement), thus causing larger poststimulus activation and more regular patterns in striatum. Reinforcement is maximal for the clinically relevant 130-Hz stimulation and restores a more normal activity in the nuclei downstream. These results suggest that reinforcement may be pivotal to achieve pattern regularization and restore the neural activity in the nuclei downstream and may stem from frequency-selective resonant properties of the loop.High-frequency (i.e., above 100 Hz) deep brain stimulation (HFS) of the basal ganglia (BG) and thalamus is clinically recognized to treat movement disorders in Parkinson’s disease (PD) (1–4), but its therapeutic mechanisms remain unclear (5, 6).Early hypotheses about HFS were derived from the rate-based model of the BG function (7, 8) and postulated the disruption of the output of the BG-thalamic system via either the inactivation of neurons in the stimulated site (target) (9–15), which would provide an effect similar to a surgical lesion, or the abnormal excitation of axons projecting out of the target (16–19), which would disrupt the neuronal activity in the structures downstream, including any pathophysiological activity (20).More recently, an ever-growing number of experiments in PD humans and animal models of Parkinsonism has indicated that HFS affects the firing patterns of the neurons rather than the mean firing rate both in the target and the structures downstream (18, 19, 21–31) and it replaces repetitive low-frequency (i.e., ≤50 Hz) bursting patterns with regularized (i.e., more tonic) patterns at higher frequencies (25, 26). It has been proposed that increased pattern regularity of neurons in the target may be therapeutic (5, 32–37), but it is still unknown how this regularity comes about with HFS.It has been suggested that an increased pattern regularity can deplete the information content of the target output and this lack of information would act as an “information lesion” (33) and prevent the pathological activity from being transmitted within the BG-thalamic system (22, 33, 36). As a result, an information lesion in the target [typically, one among the subthalamic nucleus (STN), internal globus pallidus (GPi), or thalamus] would have effects similar to those of a destructive lesion in the same site, which has been reported to alleviate the movement disorders (38).Instead, studies (32, 34, 35, 37) have suggested that an increased pattern regularity of the BG output partly compensates the PD-evoked impairment of the information-processing capabilities of the thalamo-cortical system, and this restores a more faithful thalamic relay of the sensorimotor information (35, 39).Although intriguing, these hypotheses remain elusive on (i) the neuronal mechanisms that would elicit pattern regularization (e.g., why regularization would be relevant only for HFS) and (ii) the effects that increased regularity would have on the cortico-BG-thalamo-cortical loop.It has been hypothesized that pattern regularization occurs because axons projecting out of the target follow the pattern of the stimulus pulses (40, 41) and, given the segregated organization of the BG-thalamic connections (42), it has been assumed that pattern regularization percolates straightforward from the target to the structures immediately downstream (34, 36). However, this representation of the pattern regularization as a “local” effect can hardly be reconciled with the fact that HFS of any structure of the cortico-BG-thalamo-cortical loop is therapeutic for at least some movement disorders (1–4, 43–47), nor does it explain why stimulation at frequencies above 160–180 Hz is not necessarily therapeutic despite the fact that the regularity of the axonal patterns may increase (48, 49). Moreover, coherence in the 8–30-Hz band among neurons across different structures may decrease under HFS but not for lower frequencies (26, 50–52), which suggests the emergence of diffused changes in neuronal activity that would be hardly accounted for with purely local effects.There is emerging evidence, instead, that HFS affects multiple structures simultaneously. First, it has been shown that deep brain stimulation (DBS) may antidromically activate afferent axons and fibers of passage (53–59), thus reaching structures not immediately downstream. Second, studies (57, 58) observed in 6-hydroxydopamine (6-OHDA)-intoxicated rats that the antidromic effects increase with the stimulation frequency and peak around 110–130 Hz. Third, it has been shown in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-intoxicated nonhuman primates (NHPs) that STN DBS may evoke similar poststimulus responses in different BG structures, both downstream from and upstream to the STN (5, 27, 28, 30, 60). Finally, it has been reported that the cortico-BG-thalamo-cortical system consists of multiple sets of reentrant, interconnected, and partially overlapping neuronal loops (5, 42, 61, 62), which means that the structures upstream to the target (e.g., the striatum) may play an important role in the therapeutic mechanisms of HFS.Altogether, these results suggest that (A) pattern regularization is a global effect that exploits the closed-loop nature of the cortico-BG-thalamo-cortical system and selectively emerges only for specific HFS values, and that (B) the therapeutic merit of pattern regularization has to deal with the restoration of a more normal functionality of the entire cortico-BG-thalamo-cortical loop rather than with variations in the information content of one specific structure.We explored hypotheses (A) and (B) and assessed the system-wide effects of DBS by constructing a computational model of the cortico-BG-thalamo-cortical loop in both normal and parkinsonian conditions and by simulating the effects of STN DBS both at low (20–80 Hz) and high (100–180 Hz) frequencies. The model includes populations of single-compartment neurons and interneurons from motor cortex, striatum, GPi, and thalamus according to a network topology derived from the NHP anatomy, and it simulates both the orthodromic and antidromic effects of DBS. As a result, this model reproduced both average activity and discharge patterns of single units in NHP and rats under normal and parkinsonian conditions, with and without DBS, for all modeled structures.We show through numerical simulation that hypothesis (A) is significantly contributed by reinforcement mechanisms in the striatum. These mechanisms are selectively elicited by HFS, facilitate the percolation of regularized discharge patterns from the striatum to the GPi, and have a primary role in (B), because the percolated striato-pallidal input combines with the local effects of STN DBS to restore the thalamic relay function (63).     

Dopamine release from transplanted neural stem cells in Parkinsonian rat striatum in vivo

Embryonic stem cell-based therapies exhibit great potential for the treatment of Parkinson’s disease (PD) because they can significantly rescue PD-like behaviors. However, whether the transplanted cells themselves release dopamine in vivo remains elusive. We and others have recently induced human embryonic stem cells into primitive neural stem cells (pNSCs) that are self-renewable for massive/transplantable production and can efficiently differentiate into dopamine-like neurons (pNSC–DAn) in culture. Here, we showed that after the striatal transplantation of pNSC–DAn, (i) pNSC–DAn retained tyrosine hydroxylase expression and reduced PD-like asymmetric rotation; (ii) depolarization-evoked dopamine release and reuptake were significantly rescued in the striatum both in vitro (brain slices) and in vivo, as determined jointly by microdialysis-based HPLC and electrochemical carbon fiber electrodes; and (iii) the rescued dopamine was released directly from the grafted pNSC–DAn (and not from injured original cells). Thus, pNSC–DAn grafts release and reuptake dopamine in the striatum in vivo and alleviate PD symptoms in rats, providing proof-of-concept for human clinical translation.Parkinson’s disease (PD) is a chronic progressive neurodegenerative disorder characterized by the specific loss of dopaminergic neurons in the substantia nigra pars compacta and their projecting axons, resulting in loss of dopamine (DA) release in the striatum (1). During the last two decades, cell-replacement therapy has proven, at least experimentally, to be a potential treatment for PD patients (2–7) and in animal models (8–15). The basic principle of cell therapy is to restore the DA release by transplanting new DA-like cells. Until recently, obtaining enough transplantable cells was a major bottleneck in the practicability of cell therapy for PD. One possible source is embryonic stem cells (ESCs), which can develop infinitely into self-renewable pluripotent cells with the potential to generate any type of cell, including DA neurons (DAns) (16, 17).Recently, several groups including us have introduced rapid and efficient ways to generate primitive neural stem cells (pNSCs) from human ESCs using small-molecule inhibitors under chemically defined conditions (12, 18, 19). These cells are nonpolarized neuroepithelia and retain plasticity upon treatment with neuronal developmental morphogens. Importantly, pNSCs differentiate into DAns (pNSC–DAn) with high efficiency (∼65%) after patterning by sonic hedgehog (SHH) and fibroblast growth factor 8 (FGF8) in vitro, providing an immediate and renewable source of DAns for PD treatment. Importantly, the striatal transplantation of human ESC-derived DA-like neurons, including pNSC–DAn, are able to relieve the motor defects in a PD rat model (11–13, 15, 19–23). Before attempting clinical translation of pNSC–DAn, however, there are two fundamental open questions. (i) Can pNSC–DAn functionally restore the striatal DA levels in vivo? (ii) What cells release the restored DA, pNSC–DAn themselves or resident neurons/cells repaired by the transplants?Regarding question 1, a recent study using nafion-coated carbon fiber electrodes (CFEs) reported that the amperometric current is rescued in vivo by ESC (pNSC–DAn-like) therapy (19). Both norepinephrine (NE) and serotonin are present in the striatum (24, 25). However, CFE amperometry/chronoamperometry alone cannot distinguish DA from other monoamines in vivo, such as NE and serotonin (Fig. S1) (see also refs. 26–28). Considering that the compounds released from grafted ESC-derived cells are unknown, the work of Kirkeby et al. was unable to determine whether DA or other monoamines are responsible for the restored amperometric signal. Thus, the key question of whether pNSC–DAn can rescue DA release needs to be reexamined for the identity of the restored amperometric signal in vivo.Regarding question 2, many studies have proposed that DA is probably released from the grafted cells (8, 12, 13, 20), whereas others have proposed that the grafted stem cells might restore striatal DA levels by rescuing injured original cells (29, 30). Thus, whether the grafted cells are actually capable of synthesizing and releasing DA in vivo must be investigated to determine the future cellular targets (residual cells versus pNSC–DAn) of treatment.To address these two mechanistic questions, advanced in vivo methods of DA identification and DA recording at high spatiotemporal resolution are required. Currently, microdialysis-based HPLC (HPLC) (31–33) and CFE amperometric recordings (34, 35) have been used independently by different laboratories to assess evoked DA release from the striatum in vivo. The major advantage of microdialysis-based HPLC is to identify the substances secreted in the cell-grafted striatum (33), but its spatiotemporal resolution is too low to distinguish the DA release site (residual cells or pNSC–DAn). In contrast, the major advantage of CFE-based amperometry is its very high temporal (ms) and spatial (μm) resolution, making it possible to distinguish the DA release site (residual cells or pNSC–DAn) in cultured cells, brain slices, and in vivo (34–39), but it is unable to distinguish between low-level endogenous oxidizable substances (DA versus serotonin and NE) in vivo.In the present study, we developed a challenging experimental paradigm of combining the two in vivo methods, microdialysis-based HPLC and CFE amperometry, to identify the evoked substance as DA and its release site as pNSC–DAn in the striatum of PD rats.     

Distinct dopamine neurons mediate reward signals for short- and long-term memories

Drosophila melanogaster can acquire a stable appetitive olfactory memory when the presentation of a sugar reward and an odor are paired. However, the neuronal mechanisms by which a single training induces long-term memory are poorly understood. Here we show that two distinct subsets of dopamine neurons in the fly brain signal reward for short-term (STM) and long-term memories (LTM). One subset induces memory that decays within several hours, whereas the other induces memory that gradually develops after training. They convey reward signals to spatially segregated synaptic domains of the mushroom body (MB), a potential site for convergence. Furthermore, we identified a single type of dopamine neuron that conveys the reward signal to restricted subdomains of the mushroom body lobes and induces long-term memory. Constant appetitive memory retention after a single training session thus comprises two memory components triggered by distinct dopamine neurons.Memory of a momentous event persists for a long time. Whereas some forms of long-term memory (LTM) require repetitive training (1–3), a highly relevant stimulus such as food or poison is sufficient to induce LTM in a single training session (4–7). Recent studies have revealed aspects of the molecular and cellular mechanisms of LTM formation induced by repetitive training (8–11), but how a single training induces a stable LTM is poorly understood (12).Appetitive olfactory learning in fruit flies is suited to address the question, as a presentation of a sugar reward paired with odor induces robust short-term memory (STM) and LTM (6, 7). Odor is represented by a sparse ensemble of the 2,000 intrinsic neurons, the Kenyon cells (13). A current working model suggests that concomitant reward signals from sugar ingestion cause associative plasticity in Kenyon cells that might underlie memory formation (14–20). A single activation session of a specific cluster of dopamine neurons (PAM neurons) by sugar ingestion can induce appetitive memory that is stable over 24 h (19), underscoring the importance of sugar reward to the fly.The mushroom body (MB) is composed of the three different cell types, α/β, α′/β′, and γ, which have distinct roles in different phases of appetitive memories (11, 21–25). Similar to midbrain dopamine neurons in mammals (26, 27), the structure and function of PAM cluster neurons are heterogeneous, and distinct dopamine neurons intersect unique segments of the MB lobes (19, 28–34). Further circuit dissection is thus crucial to identify candidate synapses that undergo associative modulation.By activating distinct subsets of PAM neurons for reward signaling, we found that short- and long-term memories are independently formed by two complementary subsets of PAM cluster dopamine neurons. Conditioning flies with nutritious and nonnutritious sugars revealed that the two subsets could represent different reinforcing properties: sweet taste and nutritional value of sugar. Constant appetitive memory retention after a single training session thus comprises two memory components triggered by distinct reward signals.     

Dissecting neural pathways for forgetting in Drosophila olfactory aversive memory

Recent studies have identified molecular pathways driving forgetting and supported the notion that forgetting is a biologically active process. The circuit mechanisms of forgetting, however, remain largely unknown. Here we report two sets of Drosophila neurons that account for the rapid forgetting of early olfactory aversive memory. We show that inactivating these neurons inhibits memory decay without altering learning, whereas activating them promotes forgetting. These neurons, including a cluster of dopaminergic neurons (PAM-β′1) and a pair of glutamatergic neurons (MBON-γ4>γ1γ2), terminate in distinct subdomains in the mushroom body and represent parallel neural pathways for regulating forgetting. Interestingly, although activity of these neurons is required for memory decay over time, they are not required for acute forgetting during reversal learning. Our results thus not only establish the presence of multiple neural pathways for forgetting in Drosophila but also suggest the existence of diverse circuit mechanisms of forgetting in different contexts.Although forgetting commonly has a negative connotation, it is a functional process that shapes memory and cognition (1–4). Recent studies, including work in relatively simple invertebrate models, have started to reveal basic biological mechanisms underlying forgetting (5–15). In Drosophila, single-session Pavlovian conditioning by pairing an odor (conditioned stimulus, CS) with electric shock (unconditioned stimulus, US) induces aversive memories that are short-lasting (16). The memory performance of fruit flies is observed to drop to a negligible level within 24 h, decaying rapidly early after training and slowing down thereafter (17). Memory decay or forgetting requires the activation of the small G protein Rac, a signaling protein involved in actin remodeling, in the mushroom body (MB) intrinsic neurons (6). These so-called Kenyon cells (KCs) are the neurons that integrate CS–US information (18, 19) and support aversive memory formation and retrieval (20–22). In addition to Rac, forgetting also requires the DAMB dopamine receptor (7), which has highly enriched expression in the MB (23). Evidence suggests that the dopamine-mediated forgetting signal is conveyed to the MB by dopamine neurons (DANs) in the protocerebral posterior lateral 1 (PPL1) cluster (7, 24). Therefore, forgetting of olfactory aversive memory in Drosophila depends on a particular set of intracellular molecular pathways within KCs, involving Rac, DAMB, and possibly others (25), and also receives modulation from extrinsic neurons. Although important cellular evidence supporting the hypothesis that memory traces are erased under these circumstances is still lacking, these findings lend support to the notion that forgetting is an active, biologically regulated process (17, 26).Although existing studies point to the MB circuit as essential for forgetting, several questions remain to be answered. First, whereas the molecular pathways for learning and forgetting of olfactory aversive memory are distinct and separable (6, 7), the neural circuits seem to overlap. Rac-mediated forgetting has been localized to a large population of KCs (6), including the γ-subset, which is also critical for initial memory formation (21, 27). The site of action of DAMB for forgetting has yet to be established; however, the subgroups of PPL1-DANs implicated in forgetting are the same as those that signal aversive reinforcement and are required for learning (28–30). It leaves open the question of whether the brain circuitry underlying forgetting and learning is dissociable, or whether forgetting and learning share the same circuit but are driven by distinct activity patterns and molecular machinery (26). Second, shock reinforcement elicits multiple memory traces through at least three dopamine pathways to different subdomains in the MB lobes (28, 29). Functional imaging studies have also revealed Ca²⁺-based memory traces in different KC populations (31). It is poorly understood how forgetting of these memory traces differs, and it remains unknown whether there are multiple regulatory neural pathways. Notably, when PPL1-DANs are inactivated, forgetting still occurs, albeit at a lower rate (7). This incomplete block suggests the existence of an additional pathway(s) that conveys forgetting signals to the MB. Third, other than memory decay over time, forgetting is also observed through interference (32, 33), when new learning or reversal learning is introduced after training (6, 34, 35). Time-based and interference-based forgetting shares a similar dependence on Rac and DAMB (6, 7). However, it is not known whether distinct circuits underlie forgetting in these different contexts.In the current study, we focus on the diverse set of MB extrinsic neurons (MBENs) that interconnect the MB lobes with other brain regions, which include 34 MB output neurons (MBONs) of 21 types and ∼130 dopaminergic neurons of 20 types in the PPL1 and protocerebral anterior medial (PAM) clusters (36, 37). These neurons have been intensively studied in olfactory memory formation, consolidation, and retrieval in recent years (e.g., 24, 28–30, 38–48); however, their roles in forgetting have not been characterized except for the aforementioned PPL1-DANs. In a functional screen, we unexpectedly found that several Gal4 driver lines of MBENs showed significantly better 3-h memory retention when the Gal4-expressing cells were inactivated. The screen has thus led us to identify two types of MBENs that are not involved in initial learning but play important and additive roles in mediating memory decay. Furthermore, neither of these MBEN types is required for reversal learning, supporting the notion that there is a diversity of neural circuits that drive different forms of forgetting.     

Quantum mechanical calculations suggest that lytic polysaccharide monooxygenases use a copper-oxyl,oxygen-rebound mechanism

Lytic polysaccharide monooxygenases (LPMOs) exhibit a mononuclear copper-containing active site and use dioxygen and a reducing agent to oxidatively cleave glycosidic linkages in polysaccharides. LPMOs represent a unique paradigm in carbohydrate turnover and exhibit synergy with hydrolytic enzymes in biomass depolymerization. To date, several features of copper binding to LPMOs have been elucidated, but the identity of the reactive oxygen species and the key steps in the oxidative mechanism have not been elucidated. Here, density functional theory calculations are used with an enzyme active site model to identify the reactive oxygen species and compare two hypothesized reaction pathways in LPMOs for hydrogen abstraction and polysaccharide hydroxylation; namely, a mechanism that employs a η¹-superoxo intermediate, which abstracts a substrate hydrogen and a hydroperoxo species is responsible for substrate hydroxylation, and a mechanism wherein a copper-oxyl radical abstracts a hydrogen and subsequently hydroxylates the substrate via an oxygen-rebound mechanism. The results predict that oxygen binds end-on (η¹) to copper, and that a copper-oxyl–mediated, oxygen-rebound mechanism is energetically preferred. The N-terminal histidine methylation is also examined, which is thought to modify the structure and reactivity of the enzyme. Density functional theory calculations suggest that this posttranslational modification has only a minor effect on the LPMO active site structure or reactivity for the examined steps. Overall, this study suggests the steps in the LPMO mechanism for oxidative cleavage of glycosidic bonds.Carbohydrates are the most diverse set of biomolecules, and thus, many enzyme classes have evolved to assemble, modify, and depolymerize carbohydrates, including glycosyltransferases, glycoside hydrolases, carbohydrate esterases, and polysaccharide lyases (1). Recently, a new enzymatic paradigm was discovered that employs copper-dependent oxidation to cleave glycosidic bonds in polysaccharides (2–13). These newly classified enzymes, termed lytic polysaccharide monooxygenases (LPMOs), broadly resemble other copper monooxygenases and some hydroxylation catalysts (14–21).The discovery that LPMOs use an oxidative mechanism has attracted interest both because it is a unique paradigm for carbohydrate modification that employs a powerful C–H activation mechanism, and also because LPMOs synergize with hydrolytic enzymes in biomass conversion to sugars because they act directly on the crystalline polysaccharide surface without the requirement for depolymerization (4, 22, 23), making them of interest in biofuels production. LPMOs were originally characterized as Family 61 glycoside hydrolases (GH61s, reclassified as auxiliary activity 9, AA9) or Family 33 carbohydrate-binding modules (CBM33s, reclassified as AA10), which are structurally similar enzymes found in fungi and nonfungal organisms (22), respectively. In 2005, Vaaje-Kolstad et al. described the synergism (24) of a chitin-active CBM33 (chitin-binding protein, CBP21) with hydrolases, but the mechanism was not apparent. Harris et al. demonstrated that a GH61 boosts hydrolytic enzyme activity on lignocellulosic biomass (2). Vaaje-Kolstad et al. subsequently showed that CBP21 employs an oxidative mechanism to cleave glycosidic linkages in chitin (4).Following these initial discoveries, multiple features of LPMOs have been elucidated. LPMOs use copper (5–7) and produce either aldonic acids or 4-keto sugars at oxidized chain ends, believed to result from hydroxylation at the C1 or C4 carbon, respectively. Hydroxylation at the C1 carbon is proposed to spontaneously undergo elimination to a lactone followed by hydrolytic ring opening to an aldonic acid, whereas hydroxylation and elimination at C4 yields a 4-keto sugar at the nonreducing end (5–12). The active site is a mononuclear type(II) copper center ligated by a “histidine brace” (5, 12), comprising a bidentate N-terminal histidine ligand via the amino terminus and an imidazole ring nitrogen atom and another histidine residue also via a ring nitrogen atom. Hemsworth et al. reported a bacterial LPMO structure wherein the active site copper ion was photoreduced to Cu(I) (12), and Aachmann et al. demonstrated that Cu(I) binds with higher affinity than Cu(II) in CBP21 (13). A structural study of a fungal LPMO revealed an N-terminal methylation on a nitrogen atom in the imidazole ring of unknown function (5), but some LPMOs are active without this modification (6, 11). LPMOs require reducing agents for activity such as ascorbate (2–8, 10–12), and cellobiose dehydrogenase (CDH), a common fungal secretome component, can potentiate LPMO activity in lieu of a small-molecule reducing agent (7, 8).Overall, many structural and mechanistic insights have been reported since the discoveries that LPMOs are oxidative enzymes (4–10). However, many questions remain regarding LPMO function (22, 25). Here, we examine the LPMO catalytic mechanism with density functional theory (DFT) calculations on an active site model (ASM) of a fungal LPMO. We seek to (i) understand the identity of the reactive oxygen species (ROS), (ii) compare two hypothesized catalytic mechanisms, and (iii) examine the role of N-terminal methylation in catalysis.     

From the Cover: Mother’s voice and heartbeat sounds elicit auditory plasticity in the human brain before full gestation

Brain development is largely shaped by early sensory experience. However, it is currently unknown whether, how early, and to what extent the newborn’s brain is shaped by exposure to maternal sounds when the brain is most sensitive to early life programming. The present study examined this question in 40 infants born extremely prematurely (between 25- and 32-wk gestation) in the first month of life. Newborns were randomized to receive auditory enrichment in the form of audio recordings of maternal sounds (including their mother’s voice and heartbeat) or routine exposure to hospital environmental noise. The groups were otherwise medically and demographically comparable. Cranial ultrasonography measurements were obtained at 30 ± 3 d of life. Results show that newborns exposed to maternal sounds had a significantly larger auditory cortex (AC) bilaterally compared with control newborns receiving standard care. The magnitude of the right and left AC thickness was significantly correlated with gestational age but not with the duration of sound exposure. Measurements of head circumference and the widths of the frontal horn (FH) and the corpus callosum (CC) were not significantly different between the two groups. This study provides evidence for experience-dependent plasticity in the primary AC before the brain has reached full-term maturation. Our results demonstrate that despite the immaturity of the auditory pathways, the AC is more adaptive to maternal sounds than environmental noise. Further studies are needed to better understand the neural processes underlying this early brain plasticity and its functional implications for future hearing and language development.One of the first acoustic stimuli we are exposed to before birth is the voice of the mother and the sounds of her heartbeat. As fetuses, we have substantial capacity for auditory learning and memory already in utero (1–5), and we are particularly tuned to acoustic cues from our mother (6–9). Previous research suggests that the innate preference for mother’s voice shapes the developmental trajectory of the brain (10, 11). Prenatal exposure to mother’s voice may therefore provide the brain with the auditory fitness necessary to process and store speech information immediately after birth (12, 13).There is evidence to suggest that prenatal exposure to the maternal voice and heartbeat sounds can pave the neural pathways in the brain for subsequent development of hearing and language skills (14). For example, the periodic perception of the low-frequency maternal heartbeat in the womb provides the fetus with an important rhythmic experience (15, 16) that likely establishes the neural basis for auditory entrainment and synchrony skills necessary for vocal, gestural, and gaze communication during mother–infant interactions (17, 18).Studies examining the neural response to the maternal voice soon after birth have found activation in posterior temporal regions, preferentially on the left side, as well as brain areas involved in emotional processing including the amygdala and orbito-frontal cortex (19). Similarly, Beauchemin et al. have found activation in language-related cortical regions when newborns listened to their mother’s voice, whereas a stranger’s voice seemed to activate more generic regions of the brain (20). In addition, Partanen et al. have shown that the neural response to maternal sounds depends on experience as full-term newborns react differentially to familiar vs. unfamiliar sounds they were exposed to as fetuses, suggesting correlation between the amount of prenatal exposure and brain activity (21). Taken together, the above studies suggest that the mother’s voice plays a special role in the early shaping of auditory and language areas of the brain.Numerous animal studies have shown that brain development relies on developmentally appropriate acoustic stimulation early in life (22–32). Auditory deprivation during critical periods can adversely affect brain maturation and lead to long-lasting neural despecialization in the auditory cortex (AC), whereas auditory enrichment in the early postnatal period can enhance neural sensitivity in the primary AC, as well as improve auditory recognition and discrimination abilities.Preterm infants are born during a critical period for auditory brain development. However, the maternal auditory nursery provided by the womb vanishes after a premature birth as the preterm newborn enters the neonatal intensive care unit (NICU). The abrupt transition of the fetus from the protected environment of the womb to the exposed environment of the hospital imposes significant challenges on the developing brain (33). These challenges have been associated with neuropathologic consequences, including reduction in regional brain volumes, white matter microstructural abnormalities, and poor cognitive and language outcomes in preterm compared with full-term newborns (34–41).Considering the acoustic gap between the NICU environment and the womb, it is not surprising that auditory brain development is compromised in preterm compared with full-term infants (42, 43). Numerous studies have suggested that the auditory environment available for preterm infants in the NICU may not be conducive for their neurodevelopment (44–47). These concerns are derived from the frequent reality that hospitalized preterm newborns are overexposed to loud, toxic, and unpredictable environmental noise generated by ventilators, infusion pumps, fans, telephones, pagers, monitors, and alarms (48–51), whereas at the same time they are also deprived of the low-frequency, patterned, and biologically familiar sounds of their mother’s voice and heartbeat, which they would otherwise be hearing in utero (33, 45). In addition, the hospital environment contains a significant amount of high-frequency electronic sounds (52, 53) that are less likely to be heard in the womb because of the sound attenuation provided by maternal tissues and fluid within the intrauterine cavity (54–56). Efforts to improve the hospital environment for preterm neonates have primarily focused on reducing hospital noise and maintaining a quiet environment. However, exposing medically fragile preterm newborns to low-frequency audio recordings of their mothers on a daily basis has been less acknowledged to be of necessity, and the extent to which such maternal sound exposure can influence brain maturation after an extremely premature birth has been a matter of much debate.The present study aimed to determine whether enriching the auditory environment for preterm newborns with authentic recordings of their mother’s voice and heartbeat sounds in the first month of life would result in structural alterations in the AC. The rationale driving this question lies in the fact that such enriched maternal sound stimulation would otherwise be present had the baby not been born prematurely.