Biology subjects

Hartmann, E.

Publications and source records attributed to Hartmann, E..

12 recordsLinked to original sources

bioRxiv2026

Structural basis of the two-photon photoactivation mechanism of orange carotenoid protein

Cyanobacteria have produced Earths oxygen for 2.4 billion years by adapting to fluctuating irradiance. This adaptation relies on orange carotenoid protein (OCP), which mediates light-intensity- dependent photoprotective energy dissipation using a unique two-photon absorption mechanism. Photon absorption by ground-state OCP (OCPO) generates a metastable intermediate (OCP1h{nu}) that either relaxes thermally or, upon absorption of a second photon within [~]1 s, converts to the active photoprotective state (OCPR). By integrating static and time-resolved crystallography, cryo-EM, computation, spectroscopy and biochemistry, we assign the structure of OCP1h{nu}, establish its functional relevance and capture structural snapshots along the OCPO[->]OCP1h{nu} and OCP1h{nu}[->]OCPR photochemical pathways. We elucidate the molecular mechanism of OCP, which serves as a unique biological circuit breaker protecting the photosynthetic machinery from high light flux.

biochemistry↗

bioRxiv2025

KIMMDY: a biomolecular reaction emulator

Molecular simulations have become indispensable in biological research. Their accuracy continues to improve, but directly modelling biochemical reactions - central to all life processes - remains computationally challenging. Here, we present a biomolecular reaction emulator that models reactions across conformational ensembles using kinetic Monte Carlo. Our method, KIMMDY, is capable of handling dynamic, large-scale systems with successive, competing reactions, even on the second timescale or slower. It leverages graph neural networks for large-scale prediction of reaction rates, while also being capable of using simpler physics-based or heuristic models. We validate our approach against experimental data and showcase its power and versatility through a series of applications, including radical reactions, nucleophilic substitutions, and photodimerization. Example systems span proteins and DNA. KIMMDY aids the understanding of biochemical reaction cascades in complex systems, helps to re-interpret experimental data, and can inspire future wet-lab experiments.

biochemistry↗

bioRxiv2025

Sec61 translocon inhibitor Flavitransin blocks selectively dengue virus polyprotein insertion into the ER membrane with pan-flavivirus antiviral potency

Flavivirus infections by Dengue and Zika virus impose a significant healthcare threat worldwide. At present no FDA-approved specific antiviral treatment is available, and the safety of a vaccine against Dengue virus is still under debate. Here, we report the identification of the CADA derivative flavitransin (FT), with potent activity against DENV serotype 2 in various cell types. Moreover, FT showed consistent anti-flaviviral activity against all four DENV serotypes, and also against Zika and Yellow fever virus. Viral polyprotein biogenesis was completely abolished by FT treatment of DENV-infected cells. Drug profiling by a-time-of-drug-addition assay revealed a post-entry antiviral effect of FT, in line with its anticipated Sec61 inhibitory effect. Subsequent analysis of the individual viral proteins in transfected HEK293T cells indicated that FT suppresses the expression of the structural proteins (pre-membrane and envelope) only. Furthermore, cell free in vitro protein translation analysis demonstrated a direct inhibitory effect of FT on the co-translational translocation of the DENV polyprotein across the ER membrane. More specifically, FT inhibited the initiation of protein translocation into the ER that relies on the N-terminal transmembrane region of the capsid subunit of the DENV polyprotein, resulting in rerouting of the viral pre-protein to the cytosol for proteasomal degradation. Finally, selection and genotyping of FT-resistant HCT116 cells revealed a unique A70V mutation in the Sec61 subunit that conferred resistance to FT in infected cells. Long-term exposure of DENV to FT demonstrated a high barrier to resistance development. In conclusion, our data demonstrate that FT selectively interferes with the initiation of ER co-translational translocation of the DENV polyprotein and confirm the critical role of this translocation process in the flavivirus replication cycle.

microbiology↗

bioRxiv2024

Two-photon driven photoprotection mechanism in echinenone-functionalized Orange Carotenoid Protein

Orange carotenoid protein (OCP) is a photoactive protein that mediates photoprotection in cyanobacteria. OCP binds different ketocarotenoid chromophores such as echinenone (ECN), 3- hydroxyechinenone (hECN) and canthaxanthin (CAN). In the dark, OCP is in an inactive orange form known as OCPO; upon illumination, a red active state is formed, referred to as OCPR, that can interact with the phycobilisome. Large gaps still exist in the mechanistic understanding of the events between photon absorption and formation of the OCPR state. Recent studies suggested that more than one photon may be absorbed during the photocycle. Using a two-pulse excitation setup with variable time delays we demonstrate that canthaxanthin-functionalized OCPO forms the OCPR signature after absorption of a single photon. By contrast, OCPO complexed with hECN or ECN does not photoconvert to OCPR upon single photon absorption. Instead, OCPR is formed only upon absorption of a second photon, arriving roughly one second after the first one, implying the existence of a metastable light-sensitive OCP1hv intermediate. To the best of our knowledge, a sequential 2-photon absorption mechanism in a single biological photoreceptor chromophore is unique. It results in a non-linear response function with respect to light intensity, effectively generating a threshold switch. In the case of OCP, this prevents down regulation of photosynthesis at low light irradiance.

biophysics↗

bioRxiv2024

Normal male fertility in a mouse model of KPNA2 deficiency

The nuclear transport of proteins is mediated by karyopherins and has been implicated to be crucial for germ cell and embryonic development. Deletion of distinct members of the karyopherin alpha family has been shown to cause male and female infertility in mice. Using a genetrap approach, we established mice deficient for KPNA2 (KPNA2 KO) and investigated the role of this protein in male germ cell development and fertility. Breeding of male KPNA2 KO mice leads to healthy offsprings in all cases albeit the absence of KPNA2 resulted in a reduction in sperm number by 60%. Analyses of the KPNA2 expression in wild-type mice revealed a strong KPNA2 presence in meiotic germ cells of all stages while a rapid decline is found in round spermatids. The high KPNA2 expression throughout all meiotic stages of sperm development suggests a possible function of KPNA2 during this phase, hence in its absence the spermatogenesis is not completely blocked. In KPNA2 KO mice, a higher portion of sperms presented with morphological abnormalities in the head and neck region, but a severe spermiogenesis defect was not found. Thus, we conclude that the function of KPNA2 in round spermatids is dispensable, as our mice do not show any signs of infertility. Our data provide evidence that KPNA2 is not crucial for male germ cell development and fertility.

developmental biology↗

bioRxiv2023

Karyopherin α2 is a maternal effect gene required for early embryonic development and female fertility in mice

The nuclear transport of proteins plays an important role in mediating the transition from egg to embryo and distinct karyopherins have been implicated in this process. Here, we studied the impact of KPNA2 deficiency on preimplantation embryo development in mice. Loss of KPNA2 results in complete arrest at the 2cell stage and embryos exhibit the inability to activate their embryonic genome as well as a severely disturbed nuclear translocation of Nucleoplasmin 2. Our findings define KPNA2 as a new maternal effect gene.

developmental biology↗

bioRxiv2022

Influence of pump laser fluence on ultrafast structural changes in myoglobin

High-intensity femtosecond pulses from an X-ray free-electron laser enable pump probe experiments for investigating electronic and nuclear changes during light-induced reactions. On time scales ranging from femtoseconds to milliseconds and for a variety of biological systems, time-resolved serial femtosecond crystallography (TR-SFX) has provided detailed structural data for light-induced isomerization, breakage or formation of chemical bonds and electron transfer1. However, all ultra-fast TR-SFX studies to date have employed such high pump laser energies that several photons were nominally absorbed per chromophore2-14. As multiphoton absorption may force the protein response into nonphysiological pathways, it is of great concern15 whether this experimental approach16 allows valid inferences to be drawn vis-a-vis biologically relevant single-photon-induced reactions17. Here we describe ultrafast pump-probe SFX experiments on photodissociation of carboxymyoglobin, showing that different pump laser fluences yield markedly different results. In particular, the dynamics of structural changes and observed indicators of the mechanistically important coherent oscillations of the Fe-CO bond distance (predicted by recent quantum wavepacket dynamics15) are seen to depend strongly on pump laser energy. Our results confirm both the feasibility and necessity of performing TR-SFX pump probe experiments in the linear photoexcitation regime. We consider this to be a starting point for reassessing design and interpretation of ultrafast TR-SFX pump probe experiments16 such that biologically relevant insight emerges.

biophysics↗

bioRxiv2022

RIG-I activation primes and trains innate antiviral immune memory

Adaptive processes of the innate immune system, known as trained immunity (TI), are critical to human health and disease, yet they have not been systematically investigated downstream of antiviral sensing. Here, we elucidate the potential of the antiviral cytosolic RNA receptor retinoic acid-inducible gene I (RIG-I) to train, prime and tolerize the innate immune system. Using a specific RIG-I agonist, we observed that repetitive stimulation enhanced interferon-stimulated gene (ISG) and pro-inflammatory cytokine induction in human primary monocytes, epithelial cells and fibroblasts and afforded non-specific antiviral protection. RNA sequencing revealed broad, cell type-specific transcriptional changes, indicative of priming of ISGs and training of the NF{kappa}B pathway, without measurable tolerization, while ATAC sequencing in monocytes demonstrated chromatin remodeling and enhanced accessibility of key transcription factor-binding motifs such as STAT1. Moreover, while STAT1 signaling was critically required, it was not sufficient to recapitulate RIG-I induced TI. Altogether, our data demonstrate that RIG-I-mediated TI promotes an immunologically alert state with important implications for host defense and the application of RIG-I ligands in anti-infective and anti-tumoral therapies. One Sentence SummaryRIG-I activation trains and primes innate immune response at the cellular level, affording non-specific immune protection by immune and non-immune cells.

immunology↗

bioRxiv2022

Structural insights into TRAP association with ribosome-Sec61 complex, and translocon inhibition by a CADA derivative

During co-translational translocation, the signal peptide of a nascent chain binds Sec61 translocon to initiate protein transport through the ER membrane. Our cryo-EM structure of ribosome-Sec61 shows binding of an ordered heterotetrameric TRranslocon-Associated Protein (TRAP) complex, in which TRAP-{gamma} is anchored at two adjacent positions of 28S rRNA and interacts with ribosomal protein L38 and Sec61/{gamma}. Four transmembrane helices (TMHs) of TRAP-{gamma} cluster with one C-terminal helix of each , {beta}, and {delta} subunits. The seven TMH bundle helps position a crescent-shaped trimeric TRAP-/{beta}/{delta} core in the ER lumen, facing the Sec61 channel. Further, our in vitro assay establishes the CADA derivative CK147 as a translocon inhibitor. A structure of ribosome-Sec61-CK147 reveals CK147 binding the channel and interacting with the plug helix from the lumenal side. The CK147-resistance mutations surround the inhibitor. These structures help in understanding the TRAP functions and provide a new Sec61 site for designing translocon inhibitors. Short SummaryCryo-EM structures reveal TRAP binding to ribosome-Sec61 complex, and CK147 inhibiting Sec61 by arresting the plug helix inside the channel.

cell biology↗

bioRxiv2022

Structure-function-dynamics relationships in the peculiar Planktothrix PCC7805 OCP1: impact of his-tagging and carotenoid type.

The orange carotenoid protein (OCP) is a photoactive protein involved in cyanobacterial photoprotection. Here, we report on the functional, spectral and structural characteristics of the peculiar Planktothrix PCC7805 OCP (Plankto-OCP). We show that this OCP variant is characterized by higher photoactivation and recovery rates, and a stronger energy-quenching activity, compared to other OCP studied thus far. We characterize the effect of the functionalizing carotenoid and of his-tagging on these reactions, and identify the time scales on which these modifications affect photoactivation. The presence of a his-tag at the C-terminus has a large influence on photoactivation, thermal recovery and PBS-fluorescence quenching, and likewise for the nature of the carotenoid that additionally affects the yield and characteristics of excited states and the ns-s dynamics of photoactivated OCP. By solving the structures of Plankto-OCP in the ECN- and CAN-functionalized states, each in two closely-related crystal forms, we further unveil the molecular breathing motions that animate Plankto-OCP at the monomer and dimer levels. We finally discuss the structural changes that could explain the peculiar properties of Plankto-OCP. HighlightsO_LIComplete functional characterization of Synechocystis and Planktothrix OCP C_LIO_LIHitherto unknown structures of ECN- and CAN-functionalized Planktothrix OCP C_LIO_LIInsights into fs-s timescale photodynamics of ECN- and CAN-functionalized Synechocystis and Planktothrix OCP C_LI

biophysics↗

bioRxiv2020

Importin α7 deficiency causes infertility in male mice by disrupting spermatogenesis

Spermatogenesis is driven by an ordered series of events, which rely on trafficking of specific proteins between nucleus and cytoplasm. The importin family of proteins mediates movement of specific cargo proteins when bound to importin {beta}. Importin genes have distinct expression patterns in mouse testis, implying they may have unique roles during mammalian spermatogenesis. Here we use a loss-of-function approach to specifically determine the role of importin 7 in spermatogenesis and male fertility. We show that ablation of importin 7 in male mice leads to infertility and has multiple cumulative effects on both germ cells and Sertoli cells. Importin 7-deficient mice exhibit an impaired Sertoli cell function, including loss of Sertoli cells and a compromised nuclear transport of the androgen receptor. Furthermore, our data demonstrate devastating defects in spermiogenesis that are accompanied by disturbed histone-protamine-exchange, absence of the transcriptional regulator Brwd1 and altered expression of Rfx2 target genes, resulting in incomplete sperm maturation and massive loss of sperms. Our work uncovers the essential role of importin 7 in spermatogenesis and hence in male fertility.

developmental biology↗

bioRxiv2017

DNA copy number gains of TCF4 (E2-2) are associated with poor outcome and the activated B-cell-like subtype of diffuse large B-cell lymphoma

The activated B-cell (ABC) subtype of diffuse large B-cell lymphoma (DLBCL) is characterized by the chronic activation of signaling initiated by immunoglobulin- (IgM). By analyzing DNA copy profiles of 1,000 DLBCLs, we identified gains of 18q21.2 as the most frequent genetic alteration in ABC-like DLBCL. We show that these alterations target the TCF4 (E2-2) transcription factor, and that over-expression of TCF4 leads to its occupancy on immunoglobulin gene enhancers and increased expression of IgM at the transcript and protein level. The TCF4 gene is one of the top BRD4-regulated genes in DLBCL. Using a BET proteolysis-targeting chimera (PROTAC) we show that TCF4 and IgM expression can be extinguished, and ABC-like DLBCL cells can be killed in vitro and in vivo. This highlights a novel genetic mechanism for promoting immunoglobulin signaling in ABC-like DLBCL and provides a functional rationale for the use of BET inhibitors in this disease.

genomics↗