Biology subjects

Vollmar, L.

Publications and source records attributed to Vollmar, L..

4 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↗

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↗