Biology subjects

Wisniewska, M.

Publications and source records attributed to Wisniewska, M..

9 recordsLinked to original sources

bioRxiv2026

Injector-based pump-probe serial synchrotron crystallography of CrPhotLOV1 at the MicroMAX beamline

Time-resolved serial synchrotron crystallography (TR-SSX) has become a powerful approach for investigating structural dynamics in proteins under near-physiological conditions. Its successful application, however, depends on robust sample delivery, reliable pump-probe synchronization, and reproducible data-processing strategies. Here, an injector-based pump-probe TR-SSX workflow at the MicroMAX beamline is applied to investigate the structural dynamics of the LOV1 domain of the blue-light photoreceptor phototropin from Chlamydomonas reinhardtii (CrPhotLOV1). Room-temperature serial datasets were collected for an independently measured laser-off dark state together with four light-activated pump-probe time windows (0-350, 500-850, 1000-1350 and 1500-1850 {micro}s after a microsecond laser pulse). All four time windows and an interleaved pump-probe dark reference were recorded in a single run with the JUNGFRAU detector at 2 kHz. Because thioether adduct formation in CrPhotLOV1 is complete within about 20 {micro}s and the adduct persists for hundreds of seconds, the four delays probe the same photoadduct state and serve as independent replicates. The workflow was evaluated in terms of data quality, light contamination in the pump-probe dark reference, difference Fourier analysis, extrapolated electron-density maps and refinement of light-state models. Consistent activation levels were obtained across all time points, enabling reliable structural interpretation of the light-activated state. Difference Fourier maps and refined extrapolated models reproducibly revealed formation of the characteristic FMN-Cys57 thioether adduct together with localized rearrangements within the chromophore-binding pocket. The refined light-state structures agree closely with previously reported CrPhotLOV1 models. These results establish a robust and reproducible workflow for injector-based pump-probe TR-SSX at the MicroMAX beamline, suitable for routine time-resolved studies of light-responsive proteins.

biophysics↗

bioRxiv2026

Room-temperature light-state structure of the AsLOV2-Rac1 optogenetic actuator by sheet-on-sheet serial femtosecond crystallography

Light-oxygen-voltage (LOV) domains are blue-light sensors that make fused effector proteins optically controllable. The AsLOV2-Rac1 fusion (PA-Rac1) is one of the most widely used of these tools, yet the structural basis of its activation has been inferred largely from cryogenic structures and from solution studies, mostly of the isolated AsLOV2 domain. Here, room-temperature dark and light-state structures of AsLOV2-Rac1 at 1.8 [A] resolution are reported, determined by serial femtosecond crystallography at the SwissFEL Cristallina beamline with sheet-on-sheet (SOS) chip sample delivery. Repeated 450 nm pump-laser excitation during chip rastering accumulated the FMN-cysteine adduct in about 80 % of the molecules, giving strong Fourier difference maps. To our knowledge, this is the first light-state structure of PA-Rac1 or of any AsLOV2 fusion and the first AsLOV2 light state in which the thioether adduct is not compromised by X-ray radiolysis. Apart from the N-terminal residue and one marginal Rac1 loop residue, C displacements above 0.3 [A] between dark and light models recorded at the same temperature are confined to the chromophore environment, the D helix and the G{beta}-H{beta} and H{beta}-I{beta} loops of AsLOV2. These changes, including the Ile445 rotamer switch, are also seen in light-state structures of the isolated AsLOV2 domain, but the Asn414 rotation reported there is absent in the fusion. Comparison with the cryogenic dark-state structure also shows temperature-dependent differences that exaggerate the apparent changes. In the crystal, the GTP, the Mg2+ site and the switch regions of Rac1 show no detectable light-induced change and the J helix does not unfold, although transient or low-population states cannot be excluded. This modest response is consistent with the small difference in effector affinity between dark- and lit-state mutants in solution, with lattice contacts and an interdomain Ca2+ ion as possible further contributions.

biophysics↗

bioRxiv2026

Photoactivatable Rac1: a photosensor tilt by time-resolved crystallography and a partially open switch I when GDP-bound

The LOV2 domain of Avena sativa phototropin 1 is widely used to control proteins with light, but how its signal reaches a fused effector has not been visualized. We followed photoactivatable Rac1 by time-resolved serial crystallography at room temperature. Within 350 {micro}s, a fifth of the photosensors form the flavin-cysteine adduct and tilt as a rigid body on Rac1 about an axis through the domain interface, while the J helix stays docked and Rac1 keeps GTP and dark-state switches up to 1.85 ms. Dark crystals contain a variable GDP-bound sub-population, so light data must be referenced to interleaved dark frames. In this sub-population, switch I is only partially open, held at a Ca2+ ion that bridges LOV2 and Rac1, a conformation that can persist in simulations without the lattice. The photosensor thus first acts on Rac1 at the interface that also holds switch I in the GDP-bound state.

biophysics↗

bioRxiv2026

Covalent bond formation caught in a LOV photoreceptor

Light-oxygen-voltage (LOV) domains are blue-light photoreceptors of plants, algae and fungi, and among the most widely used tools in optogenetics. They switch on by forming a covalent thioether bond between a conserved cysteine and their flavin chromophore, in a reaction that needs a proton to cross from the cysteine to the flavin through a pocket containing essentially no water. Its mechanism has been debated for two decades1, and because the chemistry is over within a microsecond its elementary steps have stayed hidden. Here we combine 10 time-resolved serial femtosecond crystallography snapshots and infrared spectroscopy with QM/MM calculations to resolve the entire sequence of events at 1.4 [A] resolution: from excited-state distortion of the flavin ring (10-100 ps), through hydration of a surface channel (10 ns) and a single ordered water reaching the active site as the reactive cysteine shifts between its conformations (100-500 ns), to the thioether bond itself, caught half-formed at 1 {micro}s (half the molecules reacted, half still poised) and complete at 10-100 {micro}s. That water bridges the cysteine and the flavin and shuttles the proton, lowering the barrier from [~]35 to [~]15 kcal/mol and accelerating the reaction by roughly fourteen orders of magnitude (without it, the half-life would be [~]237,000 years), then departs before the bond forms. Proteins can therefore hydrate a dehydrated active site transiently and on demand to overcome otherwise prohibitive reaction barriers, a catalytic strategy that reaches well beyond photoreceptors.

biophysics↗

bioRxiv2026

The genome assembly of Sphaerospora molnari provides novel insights into the rapid evolution and diversification of unique lineage-specific gene groups in myxozoan parasites

BackgroundMyxozoans are ancient cnidarian parasites with highly derived genomes characterized by an extremely accelerated rate of nucleotide substitution, abundant orphan and lineage-specific genes without further characterization. Genome data are limited to two out of the four main evolutionary lineages, assemblies are highly fragmented and often show significant levels of host contamination. ResultsWe present a near chromosome-scale myxozoan genome, based on Oxford Nanopore-reads of Sphaerospora molnari, a member of the previously uncharacterized group of blood-feeding myxozoans, thereby addressing a key gap in the subphylums phylogeny. The haploid genome assembly spans 40.17 Mb in 40 contigs, harbors 14,957 genes and shows the smallest mitogenome of myxozoans (14,015 bp). Gene gain/gene loss analyses showed that myxozoans have small ancestral gene repertoires and show highly lineage-specific genome compositions. Using comparative analyses focusing on identifying unique but diversified gene compartments in S. molnari, we discovered taxonomically restricted protist genes related to red blood cell attachment (Plasmodium ETRAMPs) and surface protein variation (Plasmodium variant surface antigen families RIFIN and STEVOR, as well as Metamonada variant-specific surface proteins, VSPs), raising questions about their origins and evolution. A genomic trait shared between several myxozoans is the significant expansion of DNA transposable elements belonging to the mutator-like elements (MULEs), and while the simple copy-paste mechanism of these transposases may suggest frequent uncontrolled mutation, we demonstrate domestication of MULEs into transcription factors. Analyses of the gene fragments of chimeric MULEs (pack-MULES) of S. molnari show that these coincide with highly diversified gene groups in this lineage, including alien genes, suggesting MULEs as a driving force for gene evolution in myxozoans. ConclusionsParasitic lifestyle shifts drive exceptionally rapid genome evolution in myxozoans, primarily through nonadaptive mutation and gene transfer via MULEs, with adaptive refinement through MULE domestication and selection. In S. molnari, these processes underpin unique erythrocyte exploitation and immune evasion strategies essential for survival in the hosts bloodstream.

genomics↗

bioRxiv2022

The RNA-binding landscape of HAX1 protein indicates its involvement in ribosome biogenesis and translation.

HAX1 is a human protein with no known homologues or structural domains, mutations in which cause severe congenital neutropenia through mechanisms that are poorly understood. Previous studies reported RNA-binding capacity of HAX1, but the role of this binding in physiology and pathology remains unexplained. Here we report transcriptome-wide characterization of HAX1 RNA targets using RIP-seq and CRAC, indicating that HAX1 binds transcripts involved in ribosome biogenesis and rRNA processing. Using CRISPR knockouts we find that RNA targets of HAX1 partially overlap with transcripts downregulated in HAX1 KO, implying a role in mRNA stabilization. Gene ontology analysis demonstrated that genes differentially expressed in HAX1 KO (including genes involved in ribosome biogenesis and translation) are also enriched in a subset of genes whose expression correlates with HAX1 expression in four analyzed neoplasms. Functional connection to ribosome biogenesis was also demonstrated by gradient sedimentation ribosome profiles, which revealed differences in the small subunit:monosome ratio in HAX1 WT/KO. We speculate that changes in HAX1 expression may be important for the etiology of HAX1-linked diseases through dysregulation of translation.

molecular biology↗

bioRxiv2020

Simulated poaching affects connectedness and efficiency in social networks of African savanna elephants

Selective harvest, such as poaching, impacts group-living animals directly through mortality of individuals with desirable traits, and indirectly by altering the structure of their social networks. Understanding the relationship between the structural network changes and group performance in wild animals remains an outstanding problem. To address this knowledge gap, we evaluate the immediate effect of disturbance on group sociality in African savanna elephants -- an example, group-living species threatened by poaching. Drawing on static association data from one free ranging population, we constructed 100 virtual networks; performed a series of experiments poaching the oldest, socially central or random individuals; and quantified the immediate change in the theoretical indices of network connectivity and efficiency of social diffusion. Although the virtual networks never broke down, targeted elimination of the socially central conspecifics, regardless of age, decreased network connectivity and efficiency. These findings hint at the need to further study resilience by modeling network reorganization and interaction-mediated socioecological learning, empirical data permitting. Our work is unique in quantifying connectivity together with global efficiency in multiple virtual networks that represent the sociodemographic diversity of elephant populations likely found in the wild. The basic design of our simulation platform makes it adaptable for hypothesis testing about the consequences of anthropogenic disturbance or lethal management on social interactions in a variety of group-living species with limited, real-world data. Author SummaryWe consider the immediate response of animal groups to human disturbance by using the African savanna elephant as an example of a group-living species threatened by poaching. Previous research in one elephant population showed that poaching-induced mortality reduced social interaction among distantly related elephants, but not among close kin. Whether this type of resilience indicates that affected populations function similarity before and after poaching is an open problem. Understanding it is important because poaching often targets the largest and most socially and ecologically experienced group members. Drawing on empirical association data, we simulated poaching in 100 virtual elephant populations and eliminated the most senior or sociable members. Targeted poaching of sociable conspecifics was more impactful. Although it did not lead to population breakdown, it hampered theoretical features of interspecific associations that in other systems have been associated with group cohesion and the efficiency of transferring social information. Our findings suggest that further inquiry into the relationship between resilience to poaching and group performance is warranted. In addition, our simulation platform offers a generalizable basis for hypothesis testing in other social species, wild or captive, subject to exploitation by humans.

animal behavior and cognition↗

bioRxiv2020

SARS-CoV-2 spike glycoprotein vaccine candidate NVX-CoV2373 elicits immunogenicity in baboons and protection in mice

The COVID-19 pandemic continues to spread throughout the world with an urgent need for a safe and protective vaccine to effectuate herd immunity to control the spread of SARS-CoV-2. Here, we report the development of a SARS-CoV-2 subunit vaccine (NVX-CoV2373) produced from the full-length spike (S) protein, stabilized in the prefusion conformation. Purified NVX-CoV2373 S form 27.2nm nanoparticles that are thermostable and bind with high affinity to the human angiotensin-converting enzyme 2 (hACE2) receptor. In mice and baboons, low-dose NVX-CoV2373 with saponin-based Matrix-M adjuvant elicits high titer anti-S IgG that is associated with blockade of hACE2 receptor binding, virus neutralization, and protection against SARS-CoV-2 challenge in mice with no evidence of vaccine-associated enhanced respiratory disease (VAERD). NVX-CoV2373 vaccine also elicits multifunctional CD4+ and CD8+ T cells, CD4+ T follicular helper T cells (Tfh), and the generation of antigen-specific germinal center (GC) B cells in the spleen. These results support the ongoing phase 1/2 clinical evaluation of the safety and immunogenicity of NVX-CoV2327 with Matrix-M (NCT04368988).

microbiology↗

bioRxiv2020

Kallikrein-related peptidase 14 activates zymogens of membrane type matrix metalloproteinases (MT-MMPs) - a CleavEx library-based analysis

Kallikrein-related peptidases (KLKs) and matrix metalloproteinases (MMPs) are secretory proteinases known to proteolytically process components of the extracellular matrix (ECM), thus modulating the pericellular environment in physiology and excessively in pathologies like cancer. However, the interconnection between these groups of proteases remains elusive. To test this hypothesis, we have developed a peptide library-based exposition system (Cleavage of exposed amino acid sequences, CleavEx) aiming at investigating the potential of KLK14 to recognize and hydrolyze proMMP sequences specifically. Initial assessment of the library identified a total of ten MMP activation domain sequences which were validated by Edman degradation. The CleavEx analysis revealed that membrane-type (MT) MMPs are likely targeted by KLK14 for activation. Correspondingly, commercially available proMT-MMPs, namely proMMP14-17 were investigated in vitro and found to be effectively processed by KLK14. Again, the expected neo-N-termini of the activated MT MMPs were yielded and confirmed by Edman degradation. In addition, the productivity of proMMP activation was analyzed by gelatin zymography, which indicated the release of fully active, mature MT-MMPs upon KLK14 treatment. Lastly, MMP14 was shown to be processed on the cell surface by KLK14 using murine fibroblasts stably overexpressing human MMP14. Herein, we propose KLK14-mediated selective activation of cell-membrane located MT-MMPs as an additional layer of their regulation within the ECM. As both, KLKs and MT-MMPs are implicated in cancer, the activation described herein may constitute an important factor in tumor progression and metastasis.

biochemistry↗