Biology subjects

Schimpf, J.

Publications and source records attributed to Schimpf, J..

8 recordsLinked to original sources

bioRxiv2026

More insights into the dynamic assembly and rotation of the archaellum motor machinery

Archaea use the archaellum for swimming motility. Rotation of the archaellum is driven by the ATPase ArlI and the membrane platform protein ArlJ. Structural studies suggest that conformational changes in ArlI power the rotation of ArlJ. However, how ATP hydrolysis is converted into rotation remains poorly under-stood. Using single-molecule total internal reflection fluorescence (TIRF) microscopy, we show that dimeric ArlJ forms a complex with ArlI. Combining conservation analysis, AlphaFold3 predictions and co-purifications, we identified conserved ArlJ residues required for interaction with ArlI, including a structural-ly rigid loop and the critical residue R341 at the ArlI interface. Motility assays in Haloferax volcanii confirmed these findings. Together, these findings support a model in which a dimeric ArlJ is interacting with ArlI via conserved residues to generate torque.

microbiology↗

bioRxiv2023

Cochaperones convey the energy of ATP hydrolysis for directional action of Hsp90

The molecular chaperone and heat shock protein Hsp90 is part of many protein complexes in eukaryotic cells. Together with its cochaperones, Hsp90 is responsible for the maturation of hundreds of clients. Although having been investigated for decades, it still is largely unknown which components are necessary for a functional complex and how the energy of ATP hydrolysis is used to enable cyclic operation. Here we use single-molecule FRET to show how cochaperones introduce directionality into Hsp90s conformational changes during its interaction with the client kinase Ste11. Most interestingly, three cochaperones are needed to couple ATP turnover to these conformational changes. All three are therefore essential for a functional cyclic operation, which requires coupling to an energy source. Finally, our findings show how the formation of sub-complexes in equilibrium followed by a directed selection of the functional complex can be the most energy efficient pathway for kinase maturation.

biophysics↗

bioRxiv2022

Morphological and genetic diversity of maize landraces along an altitudinal gradient in the Southern Andes

Maize (Zea mays ssp. mays) is a major cereal crop worldwide and is traditionally or commercially cultivated almost all over the Americas. The northwestern region of Argentina (NWA) constitutes one of the main diversity hotspots of the Southern Andes, with contrasting landscapes and a large number of landraces. Despite the extensive collections performed by the "Banco Activo de Germoplasma INTA Pergamino, Argentina" (BAP), most of them have not been characterized yet. Here we report the morphological and molecular evaluation of 30 accessions collected from NWA, along an altitudinal gradient between 1120 and 2950 meters above sea level (masl). Assessment of morphological variation in a common garden allowed the discrimination of two groups, which differed mainly in endosperm type and overall plant size. Although the groups retrieved by the molecular analyses were not consistent with morphological clusters, they showed a clear pattern of altitudinal structuring. Affinities among accessions were not in accordance with racial assignments. Overall, our results revealed that there are two maize gene pools co-existing in NWA, probably resulting from various waves of maize introduction in pre-Columbian times as well as from the adoption of modern varieties by local farmers. In conclusion, the NWA maize landraces preserved at the BAP possess high morphological and molecular variability. Our results highlight their potential as a source of diversity for increasing the genetic basis of breeding programs and provide useful information to guide future sampling and conservation efforts.

evolutionary biology↗

bioRxiv2021

Inferring kinetic rate constants from single-molecule FRET trajectories - a blind benchmark of kinetic analysis tools

Single-molecule FRET (smFRET) is a versatile technique to study the dynamics and function of biomolecules since it makes nanoscale movements detectable as fluorescence signals. The powerful ability to infer quantitative kinetic information from smFRET data is, however, complicated by experimental limitations. Diverse analysis tools have been developed to overcome these hurdles but a systematic comparison is lacking. Here, we report the results of a blind benchmark study assessing eleven analysis tools used to infer kinetic rate constants from smFRET trajectories. We tested them against simulated and experimental data containing the most prominent difficulties encountered in analyzing smFRET experiments: different noise levels, varied model complexity, non-equilibrium dynamics, and kinetic heterogeneity. Our results highlight the current strengths and limitations in inferring kinetic information from smFRET trajectories. In addition, we formulate concrete recommendations and identify key targets for future developments, aimed to advance our understanding of biomolecular dynamics through quantitative experiment-derived models.

biophysics↗

bioRxiv2021

Autophosphorylation of the KaiC-like protein ArlH inhibits oligomerisation and interaction with ArlI, the motor ATPase of the archaellum

Motile archaea are propelled by the archaellum, whose motor complex consists of the membrane protein ArlJ, the ATPase ArlI, and the ATP-binding protein ArlH. Despite its essential function and the existence of structural and biochemical data on ArlH, the role of ArlH in archaellum assembly and function remains elusive. ArlH is a structural homolog of KaiC, the central component of the cyanobacterial circadian clock. Similar to KaiC, ArlH exhibits autophosphorylation activity, which was observed for both ArlH of the euryarchaeon Pyrococcus furiosus (PfArlH) and the crenarchaeon Sulfolobus acidocaldarius (SaArlH). Using a combination of single molecule fluorescence measurements and biochemical assays, it is shown that autophosphorylation of ArlH is closely linked to the oligomeric state of ArlH bound to ArlI. These experiments also strongly suggest that ArlH is a hexamer in its functional ArlI bound state. Mutagenesis of the putative catalytic residue Glu-57 in SaArlH results in a reduced autophosphorylation activity and abolished archaellation and motility, suggesting that optimum phosphorylation activity of ArlH is essential for both archaellation and motility.

microbiology↗

bioRxiv2020

Isolated Nuclei Stiffen in Response to Low Intensity Vibration

The nucleus, central to all cellular activity, relies on both direct mechanical input and its molecular transducers to sense and respond to external stimuli. While it has been shown that isolated nuclei can adapt to applied force ex vivo, the mechanisms governing nuclear mechanoadaptation in response to physiologic forces in vivo remain unclear. To investigate nuclear mechanoadaptation in cells, we developed an atomic force microscopy (AFM) based procedure to probe live nuclei isolated from mesenchymal stem cells (MSCs) following the application of low intensity vibration (LIV) to determine whether nuclear stiffness increases as a result of LIV. Results indicated that isolated nuclei were, on average, 30% softer than nuclei tested within intact MSCs prior to LIV. When the nucleus was isolated following LIV (0.7g, 90Hz, 20min) applied four times (4x) separated by 1h intervals, stiffness of isolated nuclei increased 75% compared to non-LIV controls. LIV-induced nuclear stiffening required functional Linker of Nucleoskeleton and Cytoskeleton (LINC) complex, but was not accompanied by increased levels of the nuclear envelope proteins LaminA/C or Sun-2. While depleting LaminA/C or Sun-1&2 resulted in either a 47% or 39% increased heterochromatin to nuclear area ratio in isolated nuclei, the heterochromatin to nuclear area ratio was decreased by 25% in LIV-treated nuclei compared to controls, indicating LIV-induced changes in the chromatin structure. Overall, our findings indicate that increased apparent cell stiffness in response to exogenous mechanical challenge of MSCs in the form of LIV is in part retained by increased nuclear stiffness and changes in chromatin structure.

bioengineering↗

bioRxiv2020

Mass Photometry of Membrane Proteins

Integral membrane proteins (IMPs) are biologically highly significant but challenging to study because they require maintaining a cellular lipid-like environment. Here, we explore the application of mass photometry (MP) to IMPs and membrane mimetic systems at the single particle level. We apply MP to amphipathic vehicles, such as detergents and amphipols, as well as to lipid and native nanodiscs, characterising the particle size, sample purity and heterogeneity. Using methods established for cryogenic electron microscopy, we eliminate detergent background, enabling high-resolution studies of membrane protein structure and interactions. We find evidence that, when extracted from native membranes using native styrene-maleic acid nanodiscs, the potassium channel KcsA is present as a dimer of tetramers - in contrast to results obtained using detergent purification. Finally, using lipid nanodiscs, we show that MP can help distinguish between functional and non-functional nanodisc assemblies, as well as determine the critical factors for lipid nanodisc formation.

biophysics↗

bioRxiv2019

Quantifying the heterogeneity of macromolecular machines by mass photometry

Sample purity is central to in vitro studies of protein function and regulation, as well as to the efficiency and success of structural studies requiring crystallization or computational alignment of individual molecules. Here, we show that mass photometry (MP) accurately reports on sample heterogeneity using minimal volumes with molecular resolution within minutes. We benchmark our approach by negative stain electron microscopy (nsEM), including workflows involving chemical crosslinking and multi-step purification of a multi-subunit ubiquitin ligase. When applied to proteasome stability, we detect and quantify assemblies invisible to nsEM. Our results illustrate the unique advantages of MP for rapid sample characterization, prioritization and optimization.

biophysics↗