Biology subjects

Tosha, T.

Publications and source records attributed to Tosha, T..

10 recordsLinked to original sources

bioRxiv2026

Use of photocaged molecular oxygen for time-resolved serial X-ray crystallography

Photocages offer an attractive means of synchronously triggering enzyme-substrate driven reactions in biological systems in crystallo expanding the reach of light-driven catalysis. We describe the application of photocaged molecular oxygen to trigger molecular oxygen binding in crystals of myoglobin under anaerobic conditions and follow structural changes using both serial synchrotron and serial femtosecond X-ray crystallography. This is enabled through use of fixed targets under anaerobic conditions, utilising thin polymeric films with low molecular oxygen permeability and validated by serially collecting deoxy myoglobin structures and complementary in crystallo UV-Vis spectroscopy. Release of molecular oxygen from the photocage and subsequent binding of the gaseous ligand is structurally visualised in oxygen-bound structures of myoglobin at 5 and 10 ms and various laser parameters. We present a robust workflow for enabling anaerobic room-temperature data collection of oxygen-sensitive samples on fixed targets and report the successful photo-release of caged molecular oxygen for time-resolved serial crystallography.

molecular biology↗

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

Integrated structural dynamics uncover new modes of B12 photoreceptor activation

Photoreceptor proteins initiate, regulate and control fundamental biological processes such as vision, photosynthesis and circadian rhythms1. A large photoreceptor subfamily uses vitamin B12 derivatives for light sensing2, contrasting with the well-established mode of action of these organometallic derivatives in thermally activated enzymatic reactions3. The molecular mechanism of B12 photoreception and how this differs to the thermal pathways remain unknown. Here we provide a detailed spatio-temporal description of photoactivation in the prototypical tetrameric B12 photoreceptor CarH4,5 from nanoseconds to seconds by using an integrative approach, combining time- and temperature-resolved structural and spectroscopic methods with quantum chemical calculations. High resolution structural snapshots of key intermediates illustrate how photocleavage of a Co-C bond triggers a pathway of structural changes that propagate throughout CarH from the B12 chromophore, via a previously unknown adduct, to finally cause tetramer dissociation. These unique intermediates, which differentiate CarH from thermally-activated B12 enzymes, steer the photoactivation pathway and act as the molecular bridge between photochemical and photobiological timescales. Our results offer a spatio-temporal understanding of CarH photoactivation and pave the way for designing and optimising B12-dependent photoreceptors for future optogenetic applications.

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

Solubility, Hydration, and Sulphate Coordination in Cubic Insulin Crystals from Esrapid™ Monomers Stabilized in Divalent Anionic Form of Citric Acid

Insulins structural adaptations have been extensively studied at neutral and basic pH; however, the effects of water and anion coordination on allosteric regions under various acidic conditions remain unexplored. Given the critical role of polar interactions in allosteric modulation, investigating structured water movements across extended pH intervals is essential for understanding solvent-mediated stabilization mechanisms in the monomer form of insulin. Structures of acid-stable cubic insulin crystals were determined in the divalent anionic form of citric acid solutions over a pH range of 2 to 6 to investigate the effects of water and anion coordination, along with charge distribution, on protein conformation. Synchrotron X-ray diffraction data were collected at resolutions ranging from 1.4 to 1.76 [A], with refined models exhibiting R-factors between 0.19 and 0.21. While the spatial arrangement of most proteins is highly conserved, [~]90% of the water coordination network and polar interactions alter local residue motion and intrinsic dynamics of the structures as the pH is changed. This is in line with previously determined structures at pH 7-11, allowing for a comprehensive structural analysis in the pH range of pH 2-11. Three key observations emerged: (i) water coordinations undergo a prominent shift toward the isoelectric point of insulin (pH 5-7), (ii) water molecules and anions function as allosteric modulators to stabilize the T-state of insulin at the pH range 2 to 6, and (iii) at extreme pH values 2 and 11, increased solubility correlates with the structural adoption of insulins most active form, wherein hydration within the allosteric pocket supports monomer stabilization in the T-state. Combined with the computational analyses, pH-dependent electrostatic redistributions primarily affect side-chain dynamics and local protein motion. This solvent-coupled allosteric regulation provides a mechanistic framework for solvent-mediated protein stabilization, offering a novel insight into the rational design of insulin formulations through controlled protonation and hydration strategies.

biophysics↗

bioRxiv2024

Double crossed? Structural and computational studies of an unusual crosslinked heme in Methylococcus capsulatus cytochrome P460

Cytochromes P460 oxidise hydroxylamine within the nitrogen cycle and contain as their active site an unusual catalytic c-type heme where the porphyrin is cross-linked to the protein via a lysine residue in addition to the canonical cross links from cysteine residues. Understanding how enzymes containing P460 heme oxidise hydroxylamine into either nitrous oxide or nitric oxide has implications for climate change. Interestingly the P460 containing hydroxylamine oxidoreductase utilises a tyrosine cross link to heme and performs similar chemistry. Previous crystal structures of cytochrome P460 from Nitrosomonas europaea (NeP460) clearly show the existence of a single crosslink between the Nz atom of lysine and the heme porphyrin with mutagenesis studies indicating roles for the crosslink in positioning a proton transfer residue and/or influencing the distortion of the heme. Here we describe the evidence for a novel double cross link between lysine and heme in the cytochrome P460 from Methylococcus capsulatus (Bath). In order to understand the complexities of this enzyme system we applied high resolution structural biology approaches at synchrotron and XFEL sources paired with crystal spectroscopies. Linked to this we carried out QM/MM simulations that enabled the prediction of electronic absorption spectra providing a crucial validation to linking simulations and experimental structures. Our work demonstrates the feasibility of a double crosslink in McP460 and provides an opportunity to investigate how simulations can interact with experimental structures.

biochemistry↗

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↗

bioRxiv2024

Monomer-dimer structural comparison in quinol-dependent nitric oxide reductase reveals a functional basis for superior enzymatic activity in the dimer

The leading cause of bacterial meningitis, Neisseria meningitidis, deploys a quinol-dependent nitric oxide reductase (NmqNOR), belonging to the heme-copper oxidase superfamily. By detoxifying NO, an antimicrobial gas produced by hosts immune system, qNOR enables pathogen survival within hosts. Here, we determined cryoEM structures of the less active monomer and highly active dimer of NmqNOR at resolutions of 2.25 and 1.89 [A], respectively, showing the structural elements responsible for effective NO reduction. Helical disorder at the dimer interface, associated with an altered conformation of the critical Glu563 near the heme/non-heme Fe active site, was observed in the monomer. These findings suggest that dimerization stabilizes the active conformation of Glu563 through the structural network between the dimerization site and the active site. Since other members of the heme-copper oxidases exhibit dimerization, the current data on qNOR helps us understand a regulatory mechanism related to the function of heme-copper oxidases upon oligomerization. TeaserCryoEM structures unveil a functional rationale for dimerization in nitric oxide detoxifying enzyme from a pathogen

biochemistry↗

bioRxiv2023

A redox switch allows binding of ferrous and ferric ions in the cyanobacterial iron binding protein FutA from Prochlorococcus

The marine cyanobacterium Prochlorococcus is a main contributor to global photosynthesis, whilst being limited by iron availability. Cyanobacterial genomes typically encode two different types of FutA iron binding proteins: periplasmic FutA2 ABC transporter subunits bind Fe(III), while cytosolic FutA1 binds Fe(II). Owing to their small size and their economized genome Prochlorococcus ecotypes typically possess a single futA gene. How the encoded FutA protein might bind different Fe oxidation states was previously unknown. Here we use structural biology techniques at room temperature to probe the dynamic behavior of FutA. Neutron diffraction confirmed four negatively charged tyrosinates, that together with a neutral water molecule coordinate iron in trigonal bipyramidal geometry. Positioning of the positively charged Arg103 side chain in the second coordination shell yields an overall charge-neutral Fe(III) binding state in structures determined by neutron diffraction and serial femtosecond crystallography. Conventional rotation X-ray crystallography using a home source revealed X-ray induced photoreduction of the iron center with observation of the Fe(II) binding state; here, an additional positioning of the Arg203 side chain in the second coordination shell maintained an overall charge neutral Fe(II) binding site. Dose series using serial synchrotron crystallography and an XFEL X-ray pump-probe approach capture the transition between Fe(III) and Fe(II) states, revealing how Arg203 operates as a switch to accommodate the different iron oxidation states. This switching ability of the Prochlorococcus FutA protein may reflect ecological adaptation by genome streamlining and loss of specialized FutA proteins. Significance StatementOceanic primary production by marine cyanobacteria is a main contributor to carbon and nitrogen fixation. Prochlorococcus is the most abundant photosynthetic organism on Earth, with an annual carbon fixation comparable to the net global primary production from agriculture. Its remarkable ecological success is based on the ability to thrive in low nutrient waters. To manage iron limitation, Prochlorococcus possesses the FutA protein for iron uptake and homeostasis. We reveal a molecular switch in the FutA protein that allows it to accommodate binding of iron in either the Fe(III) or Fe(II) state using structural biology techniques at room temperature and provide a plausible mechanism for iron binding promiscuity.

biophysics↗