Structural Basis of Membrane Protein Chaperoning through the Mitochondrial Intermembrane Space.

The exchange of metabolites between the mitochondrial matrix and the cytosol depends on ß-barrel channels in the outer membrane and α-helical carrier proteins in the inner membrane. The essential translocase of the inner membrane (TIM) chaperones escort these proteins through the intermembrane space, but the structural and mechanistic details remain elusive. We have used an integrated structural biology approach to reveal the functional principle of TIM chaperones. Multiple clamp-like binding sites hold the mitochondrial membrane proteins in a translocation-competent elongated form, thus mimicking characteristics of co-translational membrane insertion. The bound preprotein undergoes conformational dynamics within the chaperone binding clefts, pointing to a multitude of dynamic local binding events. Mutations in these binding sites cause cell death or growth defects associated with impairment of carrier and ß-barrel protein biogenesis. Our work reveals how a single mitochondrial "transfer-chaperone" system is able to guide α-helical and ß-barrel membrane proteins in a "nascent chain-like" conformation through a ribosome-free compartment.

Structure and Function of the Nuclear Pore Complex Cytoplasmic mRNA Export Platform.

The last steps in mRNA export and remodeling are performed by the Nup82 complex, a large conserved assembly at the cytoplasmic face of the nuclear pore complex (NPC). By integrating diverse structural data, we have determined the molecular architecture of the native Nup82 complex at subnanometer precision. The complex consists of two compositionally identical multiprotein subunits that adopt different configurations. The Nup82 complex fits into the NPC through the outer ring Nup84 complex. Our map shows that this entire 14-MDa Nup82-Nup84 complex assembly positions the cytoplasmic mRNA export factor docking sites and messenger ribonucleoprotein (mRNP) remodeling machinery right over the NPC's central channel rather than on distal cytoplasmic filaments, as previously supposed. We suggest that this configuration efficiently captures and remodels exporting mRNP particles immediately upon reaching the cytoplasmic side of the NPC.

Superradiant Thomson scattering from graphite in the extreme ultraviolet.

We study the Thomson scattering from highly oriented pyrolitic graphite excited by the extreme ultraviolet, coherent pulses of FERMI free electron laser (FEL). An apparent nonlinear behavior is observed and fully described in terms of the coherent nature of both exciting FEL beam and scattered radiation, producing an intensity-dependent enhancement of the Thomson scattering cross-section. The process resembles Dicke's superradiant phenomenon and is thus interpreted as the observation of superradiant Thomson scattering. The process also triggers the creation of coherent, low-q ([Formula: see text] 0.3 Å[Formula: see text]), low energy phonons. The experimental data and analysis provide quantitative information on the sample characteristics, absorption, scattering factor, and coherent phonon energies and populations and open the route for the investigation of the deep nature of complex materials.

The intrinsic twinning and enigmatic twisting of aragonite crystals.

It is generally accepted that aragonite crystals of biogenic origin are characterized by significantly higher twin densities compared to samples formed during geological processes. Based on our single crystal X-ray diffraction (SCXRD) and transmission electron microscopy (TEM) study of aragonite crystals from various localities, we show that in geological aragonites, the twin densities are comparable to those of the samples from crossed lamellar zones of molluscs shells. The high twin density is consistent with performed calculations, according to which the Gibbs free energy of twin-free aragonite is close to that of periodically twinned aragonite structure. In some cases, high twin densities result in the appearance of diffuse scattering in SCXRD patterns. The obtained TEM and optical micrographs show that besides the twin boundaries (TBs) of growth origin, there are also TBs and especially stacking faults that were likely formed as the result of local strain compensation. SCXRD patterns of the samples from Tazouta, in addition to diffuse scattering lines, show Debye arcs in the [Formula: see text] plane. These Debye arcs are present only on one side of the Bragg reflections and have an azimuthal extent of nearly 30°, making the whole symmetry of the diffraction pattern distinctly chiral, which has not yet been reported for aragonite. By analogy with biogenic calcite crystals, we associate these arcs with the presence of misoriented subgrains formed as a result of crystal twisting during growth.

Orbital-selective time-domain signature of nematicity dynamics in the charge-density-wave phase of La1.65Eu0.2Sr0.15CuO4.

Understanding the interplay between charge, nematic, and structural ordering tendencies in cuprate superconductors is critical to unraveling their complex phase diagram. Using pump-probe time-resolved resonant X-ray scattering on the (0 0 1) Bragg peak at the Cu [Formula: see text] and O [Formula: see text] resonances, we investigate nonequilibrium dynamics of [Formula: see text] nematic order and its association with both charge density wave (CDW) order and lattice dynamics in La[Formula: see text]Eu[Formula: see text]Sr[Formula: see text]CuO[Formula: see text]. The orbital selectivity of the resonant X-ray scattering cross-section allows nematicity dynamics associated with the planar O 2[Formula: see text] and Cu 3[Formula: see text] states to be distinguished from the response of anisotropic lattice distortions. A direct time-domain comparison of CDW translational-symmetry breaking and nematic rotational-symmetry breaking reveals that these broken symmetries remain closely linked in the photoexcited state, consistent with the stability of CDW topological defects in the investigated pump fluence regime.

Myosin-5 varies its step length to carry cargo straight along the irregular F-actin track.

Molecular motors employ chemical energy to generate unidirectional mechanical output against a track while navigating a chaotic cellular environment, potential disorder on the track, and against Brownian motion. Nevertheless, decades of nanometer-precise optical studies suggest that myosin-5a, one of the prototypical molecular motors, takes uniform steps spanning 13 subunits (36 nm) along its F-actin track. Here, we use high-resolution interferometric scattering microscopy to reveal that myosin takes strides spanning 22 to 34 actin subunits, despite walking straight along the helical actin filament. We show that cumulative angular disorder in F-actin accounts for the observed proportion of each stride length, akin to crossing a river on variably spaced stepping stones. Electron microscopy revealed the structure of the stepping molecule. Our results indicate that both motor and track are soft materials that can adapt to function in complex cellular conditions.

Rapid single-particle chemical imaging of nanoplastics by SRS microscopy.

Plastics are now omnipresent in our daily lives. The existence of microplastics (1 µm to 5 mm in length) and possibly even nanoplastics (<1 µm) has recently raised health concerns. In particular, nanoplastics are believed to be more toxic since their smaller size renders them much more amenable, compared to microplastics, to enter the human body. However, detecting nanoplastics imposes tremendous analytical challenges on both the nano-level sensitivity and the plastic-identifying specificity, leading to a knowledge gap in this mysterious nanoworld surrounding us. To address these challenges, we developed a hyperspectral stimulated Raman scattering (SRS) imaging platform with an automated plastic identification algorithm that allows micro-nano plastic analysis at the single-particle level with high chemical specificity and throughput. We first validated the sensitivity enhancement of the narrow band of SRS to enable high-speed single nanoplastic detection below 100 nm. We then devised a data-driven spectral matching algorithm to address spectral identification challenges imposed by sensitive narrow-band hyperspectral imaging and achieve robust determination of common plastic polymers. With the established technique, we studied the micro-nano plastics from bottled water as a model system. We successfully detected and identified nanoplastics from major plastic types. Micro-nano plastics concentrations were estimated to be about 2.4 ± 1.3 × 105 particles per liter of bottled water, about 90% of which are nanoplastics. This is orders of magnitude more than the microplastic abundance reported previously in bottled water. High-throughput single-particle counting revealed extraordinary particle heterogeneity and nonorthogonality between plastic composition and morphologies; the resulting multidimensional profiling sheds light on the science of nanoplastics.

[Formula: see text]-linear resistivity from magneto-elastic scattering: Application to PdCrO2.

An electronic solid with itinerant carriers and localized magnetic moments represents a paradigmatic strongly correlated system. The electrical transport properties associated with the itinerant carriers, as they scatter off these local moments, have been scrutinized across a number of materials. Here, we analyze the transport characteristics associated with ultraclean PdCrO[Formula: see text]-a quasi-two-dimensional material consisting of alternating layers of itinerant Pd-electrons and Mott-insulating CrO[Formula: see text] layers-which shows a pronounced regime of T-linear resistivity over a wide range of intermediate temperatures. By contrasting these observations to the transport properties in a closely related material PdCoO[Formula: see text], where the CoO[Formula: see text] layers are band-insulators, we can rule out the traditional electron-phonon interactions as being responsible for this interesting regime. We propose a previously ignored electron-magneto-elastic interaction between the Pd-electrons, the Cr local moments and an out-of-plane phonon as the main scattering mechanism that leads to the significant enhancement of resistivity and a T-linear regime in PdCrO[Formula: see text] at temperatures far in excess of the magnetic ordering temperature. We suggest a number of future experiments to confirm this picture in PdCrO[Formula: see text] as well as other layered metallic/Mott-insulating materials.

Tunable nonlinear optical mapping in a multiple-scattering cavity.

Nonlinear disordered systems are not only a model system for fundamental studies but also in high demand for practical applications. However, optical nonlinearity based on intrinsic material response is weak in random scattering systems. Here, we propose and experimentally realize a highly nonlinear mapping between the scattering potential and the emerging light of a reconfigurable multiple-scattering cavity. A quantitative analysis of the degree of nonlinearity reveals its dependence on the number of scattering events. The effective order of nonlinear mapping can be tuned over a wide range at low optical lower. The strong nonlinear mapping enhances output intensity fluctuations and long-range correlations. The flexibility, robustness, and energy efficiency of our approach provides a versatile platform for exploring such nonlinear mappings for various applications.

Geometrical frustration versus Kitaev interactions in BaCo2(AsO4)2.

Recently, Co-based honeycomb magnets have been proposed as promising candidate materials to host the Kitaev spin liquid (KSL) state. One of the front-runners is BaCo2(AsO4)2 (BCAO), where it was suggested that the exchange processes between Co2+ ions via the surrounding edge-sharing oxygen octahedra could give rise to bond-dependent Kitaev interactions. In this work, we present and analyze a comprehensive inelastic neutron scattering (INS) study of BCAO with fields in the honeycomb plane. Combining the constraints from the magnon excitations in the high-field polarized state and the inelastic spin structure factor measured in zero magnetic field, we examine two leading theoretical models: the Kitaev-type [Formula: see text] model and the XXZ[Formula: see text]model. We show that the existing experimental data can be consistently accounted for by the XXZ[Formula: see text]model but not by the [Formula: see text] model, and we discuss the implications of these results for the realization of a spin liquid phase in BCAO and more generally for the realization of the Kitaev model in cobaltates.

Interfacial Cherenkov radiation from ultralow-energy electrons.

Cherenkov radiation occurs only when a charged particle moves with a velocity exceeding the phase velocity of light in that matter. This radiation mechanism creates directional light emission at a wide range of frequencies and could facilitate the development of on-chip light sources except for the hard-to-satisfy requirement for high-energy particles. Creating Cherenkov radiation from low-energy electrons that has no momentum mismatch with light in free space is still a long-standing challenge. Here, we report a mechanism to overcome this challenge by exploiting a combined effect of interfacial Cherenkov radiation and umklapp scattering, namely the constructive interference of light emission from sequential particle-interface interactions with specially designed (umklapp) momentum-shifts. We find that this combined effect is able to create the interfacial Cherenkov radiation from ultralow-energy electrons, with kinetic energies down to the electron-volt scale. Due to the umklapp scattering for the excited high-momentum Bloch modes, the resulting interfacial Cherenkov radiation is uniquely featured with spatially separated apexes for its wave cone and group cone.

Scattering evidence of positional charge correlations in polyelectrolyte complexes.

Polyelectrolyte complexation plays an important role in materials science and biology. The internal structure of the resultant polyelectrolyte complex (PEC) phase dictates properties such as physical state, response to external stimuli, and dynamics. Small-angle scattering experiments with X-rays and neutrons have revealed structural similarities between PECs and semidilute solutions of neutral polymers, where the total scattering function exhibits an Ornstein-Zernike form. In spite of consensus among different theoretical predictions, the existence of positional correlations between polyanion and polycation charges has not been confirmed experimentally. Here, we present small-angle neutron scattering profiles where the polycation scattering length density is matched to that of the solvent to extract positional correlations among anionic monomers. The polyanion scattering functions exhibit a peak at the inverse polymer screening radius of Coulomb interactions, q* ≈ 0.2 Å-1. This peak, attributed to Coulomb repulsions between the fragments of polyanions and their attractions to polycations, is even more pronounced in the calculated charge scattering function that quantifies positional correlations of all polymer charges within the PEC. Screening of electrostatic interactions by adding salt leads to the gradual disappearance of this correlation peak, and the scattering functions regain an Ornstein-Zernike form. Experimental scattering results are consistent with those calculated from the random phase approximation, a scaling analysis, and molecular simulations.

Phase separation and molecular ordering of the prion-like domain of the Arabidopsis thermosensory protein EARLY FLOWERING 3.

Liquid-liquid phase separation (LLPS) is an important mechanism enabling the dynamic compartmentalization of macromolecules, including complex polymers such as proteins and nucleic acids, and occurs as a function of the physicochemical environment. In the model plant, Arabidopsis thaliana, LLPS by the protein EARLY FLOWERING3 (ELF3) occurs in a temperature-sensitive manner and controls thermoresponsive growth. ELF3 contains a largely unstructured prion-like domain (PrLD) that acts as a driver of LLPS in vivo and in vitro. The PrLD contains a poly-glutamine (polyQ) tract, whose length varies across natural Arabidopsis accessions. Here, we use a combination of biochemical, biophysical, and structural techniques to investigate the dilute and condensed phases of the ELF3 PrLD with varying polyQ lengths. We demonstrate that the dilute phase of the ELF3 PrLD forms a monodisperse higher-order oligomer that does not depend on the presence of the polyQ sequence. This species undergoes LLPS in a pH- and temperature-sensitive manner and the polyQ region of the protein tunes the initial stages of phase separation. The liquid phase rapidly undergoes aging and forms a hydrogel as shown by fluorescence and atomic force microscopies. Furthermore, we demonstrate that the hydrogel assumes a semiordered structure as determined by small-angle X-ray scattering, electron microscopy, and X-ray diffraction. These experiments demonstrate a rich structural landscape for a PrLD protein and provide a framework to describe the structural and biophysical properties of biomolecular condensates.

Structural insights into redox signal transduction mechanisms in the control of nitrogen fixation by the NifLA system.

NifL is a conformationally dynamic flavoprotein responsible for regulating the activity of the σ54-dependent activator NifA to control the transcription of nitrogen fixation (nif) genes in response to intracellular oxygen, cellular energy, or nitrogen availability. The NifL-NifA two-component system is the master regulatory system for nitrogen fixation. NifL serves as a sensory protein, undergoing signal-dependent conformational changes that modulate its interaction with NifA, forming the NifL-NifA complex, which inhibits NifA activity in conditions unsuitable for nitrogen fixation. While NifL-NifA regulation is well understood, these conformationally flexible proteins have eluded previous attempts at structure determination. In work described here, we advance a structural model of the NifL dimer supported by a combination of scattering techniques and mass spectrometry (MS)-coupled structural analyses that report on the average structure in solution. Using a combination of small angle X-ray scattering-derived electron density maps and MS-coupled surface labeling, we investigate the conformational dynamics responsible for NifL oxygen and energy responses. Our results reveal conformational differences in the structure of NifL under reduced and oxidized conditions that provide the basis for a model for modulating NifLA complex formation in the regulation of nitrogen fixation in response to oxygen in the model diazotroph, Azotobacter vinelandii.

Identification of a conserved α-helical domain at the N terminus of human DNA methyltransferase 1.

In vertebrates, DNA methyltransferase 1 (DNMT1) contributes to preserving DNA methylation patterns, ensuring the stability and heritability of epigenetic marks important for gene expression regulation and the maintenance of cellular identity. Previous structural studies have elucidated the catalytic mechanism of DNMT1 and its specific recognition of hemimethylated DNA. Here, using solution nuclear magnetic resonance spectroscopy and small-angle X-ray scattering, we demonstrate that the N-terminal region of human DNMT1, while flexible, encompasses a conserved globular domain with a novel α-helical bundle-like fold. This work expands our understanding of the structure and dynamics of DNMT1 and provides a structural framework for future functional studies in relation with this new domain.

ASCC1 structures and bioinformatics reveal a novel helix-clasp-helix RNA-binding motif linked to a two-histidine phosphodiesterase.

Activating signal co-integrator complex 1 (ASCC1) acts with ASCC-ALKBH3 complex in alkylation damage responses. ASCC1 uniquely combines two evolutionarily ancient domains: nucleotide-binding K-Homology (KH) (associated with regulating splicing, transcriptional, and translation) and two-histidine phosphodiesterase (PDE; associated with hydrolysis of cyclic nucleotide phosphate bonds). Germline mutations link loss of ASCC1 function to spinal muscular atrophy with congenital bone fractures 2 (SMABF2). Herein analysis of The Cancer Genome Atlas (TCGA) suggests ASCC1 RNA overexpression in certain tumors correlates with poor survival, Signatures 29 and 3 mutations, and genetic instability markers. We determined crystal structures of Alvinella pompejana (Ap) ASCC1 and Human (Hs) PDE domain revealing high-resolution details and features conserved over 500 million years of evolution. Extending our understanding of the KH domain Gly-X-X-Gly sequence motif, we define a novel structural Helix-Clasp-Helix (HCH) nucleotide binding motif and show ASCC1 sequence-specific binding to CGCG-containing RNA. The V-shaped PDE nucleotide binding channel has two His-Φ-Ser/Thr-Φ (HXT) motifs (Φ being hydrophobic) positioned to initiate cyclic phosphate bond hydrolysis. A conserved atypical active-site histidine torsion angle implies a novel PDE substrate. Flexible active site loop and arginine-rich domain linker appear regulatory. Small-angle X-ray scattering (SAXS) revealed aligned KH-PDE RNA binding sites with limited flexibility in solution. Quantitative evolutionary bioinformatic analyses of disease and cancer-associated mutations support implied functional roles for RNA binding, phosphodiesterase activity, and regulation. Collective results inform ASCC1's roles in transactivation and alkylation damage responses, its targeting by structure-based inhibitors, and how ASCC1 mutations may impact inherited disease and cancer.

Fatty acid binding to the human transport proteins FABP3, FABP4, and FABP5 from a Ligand's perspective.

Fatty acid binding proteins (FABPs) are a family of amphiphilic transport proteins with high diversity in terms of their amino acid sequences and binding preferences. Beyond their main biological role as cytosolic fatty acid transporters, many aspects regarding their binding mechanism and functional specializations in human cells remain unclear. In this work, the binding properties and thermodynamics of FABP3, FABP4, and FABP5 were analyzed under various physical conditions. For this purpose, the FABPs were loaded with fatty acids bearing fluorescence or spin probes as model ligands, comparing their binding affinities via microscale thermophoresis (MST) and continuous-wave electron paramagnetic resonance (CW EPR) spectroscopy. The CW EPR spectra of non-covalently bound 5- and 16-DOXYL stearic acid (5/16-DSA) deliver in-depth information about the dynamics and chemical environments of ligands inside the binding pockets of the FABPs. EPR spectral simulations allow the construction of binding curves, revealing two different binding states ('intermediately' and 'strongly' bound). The proportion of bound 5/16-DSA depends strongly on the FABP concentration and the temperature but with remarkable differences between the three isoforms. Additionally, the more dynamic state ('intermediately bound') seems to dominate at body temperature with thermodynamic preference. The ligand binding studies were supplemented by aggregation studies via dynamic light scattering and bioinformatic analyses. Beyond the remarkably fine-tuned binding properties exhibited by each FABP, which were discernible with our EPR-centered approach, the results of this work attest to the power of simple spectroscopic experiments to provide new insights into the ligand binding mechanisms of proteins in general on a molecular level.

Recovery from desensitization in GluA2 AMPA receptors is affected by a single mutation in the N-terminal domain interface.

AMPA-type ionotropic glutamate receptors (AMPARs) are central to various neurological processes, including memory and learning. They assemble as homo- or heterotetramers of GluA1, GluA2, GluA3, and GluA4 subunits, each consisting of an N-terminal domain (NTD), a ligand-binding domain, a transmembrane domain, and a C-terminal domain. While AMPAR gating is primarily controlled by reconfiguration in the ligand-binding domain layer, our study focuses on the NTDs, which also influence gating, yet the underlying mechanism remains enigmatic. In this investigation, we employ molecular dynamics simulations to evaluate the NTD interface strength in GluA1, GluA2, and NTD mutants GluA2-H229N and GluA1-N222H. Our findings reveal that GluA1 has a significantly weaker NTD interface than GluA2. The NTD interface of GluA2 can be weakened by a single point mutation in the NTD dimer-of-dimer interface, namely H229N, which renders GluA2 more GluA1-like. Electrophysiology recordings demonstrate that this mutation also leads to slower recovery from desensitization. Moreover, we observe that lowering the pH induces more splayed NTD states and enhances desensitization in GluA2. We hypothesized that H229 was responsible for this pH sensitivity; however, GluA2-H229N was also affected by pH, meaning that H229 is not solely responsible and that protons exert their effect across multiple domains of the AMPAR. In summary, our work unveils an allosteric connection between the NTD interface strength and AMPAR desensitization.

Localization of unlabeled bepirovirsen antisense oligonucleotide in murine tissues using in situ hybridization and CARS imaging.

Current methods for detecting unlabeled antisense oligonucleotide (ASO) drugs rely on immunohistochemistry (IHC) and/or conjugated molecules, which lack sufficient sensitivity, specificity, and resolution to fully investigate their biodistribution. Our aim was to demonstrate the qualitative and quantitative distribution of unlabeled bepirovirsen, a clinical stage ASO, in livers and kidneys of dosed mice using novel staining and imaging technologies at subcellular resolution. ASOs were detected in formalin-fixed paraffin-embedded (FFPE) and frozen tissues using an automated chromogenic in situ hybridization (ISH) assay: miRNAscope. This was then combined with immunohistochemical detection of cell lineage markers. ASO distribution in hepatocytes versus nonparenchymal cell lineages was quantified using HALO AI image analysis. To complement this, hyperspectral coherent anti-Stokes Raman scattering (HS-CARS) imaging microscopy was used to specifically detect the unique cellular Raman spectral signatures following ASO treatment. Bepirovirsen was localized primarily in nonparenchymal liver cells and proximal renal tubules. Codetection of ASO with distinct cell lineage markers of liver and kidney populations aided target cell identity facilitating quantification. Positive liver signal was quantified using HALO AI, with 12.9% of the ASO localized to the hepatocytes and 87.1% in nonparenchymal cells. HS-CARS imaging specifically detected ASO fingerprints based on the unique vibrational signatures following unlabeled ASO treatment in a totally nonperturbative manner at subcellular resolution. Together, these novel detection and imaging modalities represent a significant increase in our ability to detect unlabeled ASOs in tissues, demonstrating improved levels of specificity and resolution. These methods help us understand their underlying mechanisms of action and ultimately improve the therapeutic potential of these important drugs for treating globally significant human diseases.

Submicrometer Particle Impact Dynamics and Chemistry.

Experimental studies of the collision phenomena of submicrometer particles is a developing field. This review examines the range of phenomena that can be observed with new experimental approaches. The primary focus is on single-particle impact studies enabled by charge detection mass spectrometry (CDMS) implemented using the Aerosol Impact Spectrometer (AIS) at the University of California, San Diego. The AIS combines electrospray ionization, aerodynamic lens techniques, CDMS, and an electrostatic linear accelerator to study the dynamics of particle impact over a wide range of incident velocities. The AIS has been used for single-particle impact experiments on positively charged particles of diverse composition, including polystyrene latex spheres, tin particles, and ice grains, over a wide range of impact velocities. Detection schemes based on induced charge measurements and time-of-flight mass spectrometry have enabled measurements of the impact inelasticity through the determination of the coefficient of restitution, measurements of the angular distributions of scattered submicrometer particles, and the chemical composition and dissociation of solute molecules in hypervelocity ice grain impacts.