Biology subjects

Yabashi, M.

Publications and source records attributed to Yabashi, M..

7 recordsLinked to original sources

bioRxiv2026

Serial femtosecond crystallography reveals the pH-driven allosteric mechanism of hexamer glargine

Insulin glargine is formulated at acidic pH but acts after transferring to near-neutral tissue, where its prolonged effect is commonly attributed to isoelectric depot formation. However, the structural pathway linking precipitation to delayed release has remained unresolved. Here we combine ambient-temperature serial femtosecond crystallography, solution biophysics, and multiscale network analyses to define the pH-dependent conformational landscape of hexameric glargine across pH 8.4, 7.3, 6.4, and 5.1. We resolve full hexameric glargine structures and identify a previously unreported, pH-coupled lattice transition from P1211 (near-neutral) to R3:H (acidic), accompanied by redistribution from compact phenolic Rf6-state assemblies to more plastic yet structurally coherent TRf/T3Rf3 states. This transition is accompanied by B-chain N-terminal unpeeling, phenol-pocket collapse, hydration loss, and electrostatic rewiring, and is mirrored in solution by oligomeric heterogeneity, Raman amide-I broadening, reduced thermal stability, and a blue-shifted intrinsic fluorescence maximum. Multiscale analyses further indicate that acidification does not create a new dynamical regime but reweighs pre-existing collective modes along a continuous free-energy landscape. These results support a revised mechanism in which isoelectric precipitation and delayed dissociation are mechanistically coupled through structurally organized molten-like intermediate states, linking glargine pharmacology to intrinsic allosteric redistribution within the hexamer. These findings establish a structural blueprint for benchmarking biosimilar glargine and for engineering next-generation basal insulins by tuning allosteric plasticity and intermediate-state stability.

biophysics↗

bioRxiv2026

Reciprocal-space mapping of diffuse scattering by serial femtosecond crystallography reveals analog-specific disorder in insulin analogs

Insulin detemir and insulin aspart are clinically complementary analogs engineered for distinct pharmacokinetic behavior, yet their comparative structural heterogeneity across temperature regimes remains insufficiently resolved. Here, we present a multi-scale crystallographic analysis integrating near-physiological serial femtosecond crystallography (SFX) with previously reported cryogenic and ambient multicrystal datasets for both analogs. Across conventional quality metrics, reciprocal-space intensity-field reconstructions, model-derived diffuse-scattering representations, Ramachandran stereochemical validation, solvent-accessibility coupling (SAArea-MSArea), and residue-level BDamage (a packing-normalized B-factor metric highlighting local mobility outliers) profiling, we identify a coherent ambient-versus-cryogenic contrast. Ambient datasets show broader reciprocal-space heterogeneity and more diffuse model-space distributions, consistent with increased conformational sampling outside cryogenic trapping. Despite this shared trend, disorder partitioning is analog-specific: detemir exhibits strong pseudo-translational signatures with moderate twinning, whereas aspart shows weak pseudo-translation but pronounced merohedral twinning approaching the theoretical twinned limit in ambient conditions. Importantly, backbone stereochemistry remains globally stable across all datasets, indicating that the observed differences reflect structured heterogeneity rather than model deterioration. Collectively, these findings support an ensemble-aware interpretation of insulin crystallography and provide transferable structural descriptors for analog comparison, stability assessment, and formulation-oriented design.

biophysics↗

bioRxiv2025

Compact Tape-Driven Sample Delivery System for Serial Femtosecond Crystallography

We developed a compact tape-drive with on-demand sample delivery (CoT) for time-resolved serial femtosecond crystallography (SFX) experiments that can deliver sample droplets and/or initiate reactions with a drop-on-drop strategy. Two disposable piezoelectric injectors are positioned in tandem along the tape to produce a queue of nanoliter-scale droplets. X-ray free-electron laser (XFEL) pulses arrive perpendicular to and pass through the broad face of the tape where the pulse is synchronized and aligned to the droplets and thereby enables very highly efficient SFX data collection. The tape transport speed and the delivery distance can be varied to control the mixing time from approximately 130 ms to tens of seconds. We conducted time-resolved SFX experiments utilizing a basic enzymatic reaction model of hen egg white lysozyme (HEWL) and N-acetyl-D-glucosamine (GlcNAc) to demonstrate the drop-on-drop capabilities of CoT, and the full binding process of GlcNAc to HEWL was observed at 1.3-9.7 s. SynopsisTape-driven liquid sample droplet delivery system for serial femtosecond crystallography

biophysics↗

bioRxiv2025

Deciphering the altered conformational states of bifunctional thaumarchaeal crotonyl-CoA hydratase and 3-hydroxypropionyl-CoA dehydratase from Nitrosopumilus maritimus

The thaumarchaeal 3-hydroxypropionate/4-hydroxybutyrate (3HP/4HB) cycle represents one of the most efficient mechanisms for CO2 fixation discovered to date. Within this cycle, the enzyme encoded by Nmar_1308 from Nitrosopumilus maritimus SCM1 plays a crucial role due to its dual functionality as both a crotonyl-CoA hydratase (CCAH) and a 3-hydroxypropionyl-CoA dehydratase (3HPD). Although the importance of a bifunctional enzyme for lowering the cost of biosynthesis, the details of structural dynamics are still missing. Here, in addition to our cryogenic temperature structures, we determined the first ambient temperature structures of the Nmar_1308 protein by Serial Femtosecond X-ray Crystallography (SFX). The determined structures capture previously unobserved conformational dynamics of the Nmar_1308 protein, providing invaluable information for future synthetic biology applications.

biochemistry↗

bioRxiv2025

High throughput protein serial crystallography using a grease matrix and a large-area support film

Serial femtosecond crystallography (SFX) using ultrashort pulses from X-ray free- electron lasers (XFELs) enables the determination of crystal structures at room temperature while minimizing radiation damage to the samples. This method involves irradiating numerous crystals one by one with XFEL pulses, allowing even the capture snapshots of dynamical structures in biological macromolecules. To achieve this, an efficient sample delivery system is essential for acquiring a large number of diffraction patterns. The most common approach uses a highly viscous grease matrix containing sample crystals, injected into the XFEL path from a narrow nozzle. However, the injection often suffers from clogging issues inside the injector nozzle, resulting in additional challenges such as the need for suitably sized crystals, increased sample consumption and unstable flow rates. Alternatively, a fixed-target approach, which scans a two-dimensional substrate with dispersed samples, can circumvent these issues. However, it must ensure the integrity of biological samples and provide sufficient surface area for efficient data collection. We here present an approach that utilizes a grease matrix and a large-area support film specially designed to address these requirements. This system offers a fast and reliable solution for protein SFX, enabling high-quality structure determination while significantly reducing sample consumption.

molecular biology↗

bioRxiv2024

Experimental and Computational Insights into the Structural Dynamics of the Fc Fragment of IgG1 Subtype from Biosimilar VEGF-Trap

The constant fragment (Fc) of the Immunoglobulin G1 (IgG1) subtype is increasingly recognized as a crucial scaffold in the development of advanced therapeutics due to its enhanced specificity, efficacy, and extended half-life. A prime example is VEGF-Trap (Aflibercept), a recombinant fusion protein that merges the Fc region of the IgG1 subtype with the binding domains of vascular endothelial growth factor receptors (VEGFR)-1 and VEGFR-2. The Fc regions role in N-glycosylation is particularly important, as it significantly influences protein stability. In this study, we present the first near-physiological temperature structures of the N-glycan-bound Fc fragment of IgG1 subtype from a biosimilar VEGF-Trap, determined using the SPring-8 Angstrom Compact free electron LAser (SACLA) and the Turkish Light Source (Turkish DeLight). Comparative analysis with cryogenic structures, including our own data, reveals alternate conformations within the glycan-binding pocket. Furthermore, molecular dynamics (MD) simulations highlight an unexpected degree of structural plasticity. These findings offer new insights into the molecular basis of Fc-mediated functions and provide valuable information for the design of next-generation therapeutics.

molecular biology↗

bioRxiv2017

Atomic resolution structure of serine protease proteinase K at ambient temperature

Atomic resolution structures (beyond 1.20 [A]) at ambient temperature, which is usually hampered by the radiation damage in synchrotron X-ray crystallography (SRX), will add to our understanding of the structure-function relationships of enzymes. Serial femtosecond crystallography (SFX) has attracted surging interest by providing a route to bypass such challenges. Yet the progress on atomic resolution analysis with SFX has been rather slow. In this report, we describe the 1.20 [A] resolution structure of proteinase K using 13 keV photon energy. Hydrogen atoms, water molecules, and a number of alternative side-chain conformations have been resolved. The increase in the value of B-factor in SFX suggests that the residues and water molecules adjacent to active sites were flexible and exhibited dynamic motions at specific substrate-recognition sites.

biochemistry↗