Biology subjects

Thallmair, S.

Publications and source records attributed to Thallmair, S..

9 recordsLinked to original sources

bioRxiv2026

Lipid versatility of the C2C domain of Extended Synaptotagmin 3 safeguards plasma membrane binding

Membrane contact sites between the endoplasmic reticulum (ER) and the plasma membrane (PM) are critical hubs for lipid homeostasis and signal transduction. Extended Synaptotagmins (E-Syts) tether the ER to the PM and mediate lipid transport from the PM to the ER following Ca2+ signaling. Although E-Syts are essential for maintaining ER-PM contact sites during PI(4,5)P2 resynthesis, the molecular mechanisms governing their membrane association remain poorly understood. In particular, it is unclear how these contact sites are maintained following PI(4,5)P2 hydrolysis, when their principal membrane-binding ligand is transiently depleted. Here, we characterize the membrane-binding mechanism of the C-terminal C2 domain of extended synaptotagmin 3 (E-Syt3) by combining coarse-grained molecular dynamics simulations with live-cell experiments. We identify distinct lipid binding sites with specific affinities for phosphoinositides as well as for phosphatidylinositol (PI) and phosphatidylserine (PS). While phosphoinositide interactions dominate membrane binding under resting conditions, PI and PS binding may provide an alternative membrane-binding mechanism that helps preserve ER-PM contacts during PI(4,5)P2 depletion. Our simulations further reveal that phosphoinositide binding is reinforced by the insertion of a tryptophan residue into the membrane hydrophobic core, providing a mechanistic basis for the high membrane affinity of the C2C domain. Together, our findings uncover the molecular determinants of E-Syt3 C2C-mediated membrane binding and provide mechanistic insight into the stability of E-Syt-mediated ER-PM contact sites during dynamic changes in membrane phosphoinositide composition.

molecular biology↗

bioRxiv2025

Martini 3 coarse-grained models of azobenzene-based photolipids: Modulation of membranes with light

Photoswitchable lipids offer an attractive way to manipulate the biophysical properties of membranes by means of light. Their application to modulate membrane properties, manipulate membrane proteins, and photocontrol cargo release is gaining popularity. Here, we present coarse-grained Martini 3 models for azobenzene and azobenzene-based photoswitchable lipids. Our models show good agreement with atomistic reference simulations. Furthermore, we apply our coarse-grained photolipid models to study photocontrol of lateral phase separation, protein flexibility, and membrane permeability. The results agree well with experimental data from the literature and highlight the broad applicability of our Martini 3 photolipid models. They will enable studying the impact of photolipid switching on large membrane systems as well as on their (bio)molecular interaction partners.

biophysics↗

bioRxiv2025

Photopharmacology in Action: Conformational Landscape of a Photoswitchable Covalent Kinase Inhibitor

Photopharmacology is a rapidly evolving field that uses light to control drug activity with high spatial and temporal precision, offering innovative therapeutic strategies with reduced side effects. In this study, we investigate a photoswitchable covalent inhibitor for the MAP kinase JNK3, a target for the treatment of neurodegenerative diseases. The inhibitor, which is based on a diazocine photoswitch, can undergo reversible photoisomerization of a double bond, switching between two (meta-)stable isomers. Atomistic molecular dynamics simulations, comprising almost 40 {micro}s of total simulation time, reveal how the distinct conformational spaces of the two isomers modulate their interactions with JNK3 within the ATP-binding pocket. We show that only the metastable trans isomer can form a covalent bond with JNK3, thereby permanently inhibiting its function. In contrast, for the cis state the distance between the inhibitor and the targeted cysteine residue is too large to allow covalent bond formation. Furthermore, our simulations reveal that the covalent bond, in combination with the environment of the protein pocket, hinders the full back-relaxation of the trans isomer to the stable cis isomer. Finally, our data shows that the covalently bound stable cis isomer modulates the conformations of JNK3, mainly of its activation loop. Our findings provide molecular insights into the complex dynamics of photoswitchable inhibitors, guiding future drug design for light-controlled therapeutics.

biochemistry↗

bioRxiv2025

Martini 3 protein models - a practical introduction to different structure bias models and their comparison

Biophysical characterization of protein structure and dynamics is essential in many scientific fields, including molecular biology, drug discovery, and enzyme design. Molecular dynamics (MD) simulations have become an increasingly important tool for studying these properties. This chapter provides a hands-on introduction to protein modeling with the Martini 3 coarse-grained (CG) force field. We outline its two-layer framework, where the first layer defines molecular topology and interactions, while the second layer applies structural bias to maintain secondary, tertiary, and quaternary structures. Three structure bias approaches - Elastic Network (EN), G[o]Martini, and OLIVES - are discussed, highlighting their advantages and trade-offs. Using a protein kinase as a case study, we demonstrate the step-by-step setup of Martini 3 protein models for simulations with the program package GROMACS, including system preparation, fine-tuning, and validation against atomistic reference simulations. Additionally, we explore the combination of an intrinsically disordered region (IDR) with a structured protein domain. By the end of this chapter, readers will have the necessary expertise to apply Martini 3-based protein modeling to a wide range of research applications.

biophysics↗

bioRxiv2024

GoMartini 3: From large conformational changes in proteins to environmental bias corrections

Coarse-grained modeling has become an important tool to supplement experimental measurements, allowing access to spatio-temporal scales beyond all-atom based approaches. The G[o]Martini model combines structure- and physics-based coarse-grained approaches, balancing computational efficiency and accurate representation of protein dynamics with the capabilities of studying proteins in different biological environments. This paper introduces an enhanced G[o]Martini model, which combines a virtual-site implementation of G[o] models with Martini 3. The implementation has been extensively tested by the community since the release of the new version of Martini. This work demonstrates the capabilities of the model in diverse case studies, ranging from protein-membrane binding to protein-ligand interactions and AFM force profile calculations. The model is also versatile, as it can address recent inaccuracies reported in the Martini protein model. Lastly, the paper discusses the advantages, limitations, and future perspectives of the Martini 3 protein model and its combination with G[o] models.

biophysics↗

bioRxiv2024

GPCR surface creates a favorable pathway for membrane permeation of drug molecules

G protein-coupled receptors (GPCRs) play a crucial role in modulating physiological responses and serve as the main drug target. Specifically, salmeterol and salbutamol which are used for the treatment of pulmonary diseases, exert their effects by activating the GPCR {beta}2-adrenergic receptor ({beta}2AR). In our study, we employed coarse-grained molecular dynamics simulations with the Martini 3 force field to investigate the dynamics of drug molecules in membranes in presence and absence of {beta}2AR. Our simulations reveal that in more than 50% of the flip-flop events the drug molecules use the {beta}2AR surface to permeate the membrane. The pathway along the GPCR surface is significantly more energetically favorable for the drug molecules, which was revealed by umbrella sampling simulations along spontaneous flip-flop pathways. Furthermore, we assessed the behavior of drugs with intracellular targets, such as kinase inhibitors, whose therapeutic efficacy could benefit from this observation. In summary, our results show that {beta}2AR surface interactions can significantly enhance membrane permeation of drugs, emphasizing their potential for consideration in future drug development strategies.

biophysics↗

bioRxiv2022

A Locally Activatable Sensor for Robust Quantification of Organellar Glutathione

Glutathione (GSH) is the main determinant of intracellular redox potential and participates in multiple cellular signaling pathways. Achieving a detailed understanding of intracellular GSH trafficking and regulation depends on the development of tools to map GSH compartmentalization and intra-organelle fluctuations. Herein, we present a new GSH sensing platform, TRaQ-G, for live-cell imaging. This small-molecule/protein hybrid sensor possesses a unique reactivity turn-on mechanism that ensures that the small molecule is only sensitive to GSH in the desired location. Furthermore, TRaQ-G can be fused to a fluorescent protein of choice to give a ratiometric response. Using TRaQ-G-mGold, we demonstrated that the nuclear and cytosolic GSH pools are independently regulated during cell proliferation. We also used this sensor, in combination with roGFP, to quantify redox potential and GSH concentration simultaneously in the endoplasmic reticulum. Finally, by exchanging the fluorescent protein, we created a near-infrared, targetable and quantitative GSH sensor.

cell biology↗

bioRxiv2020

Local PI(4,5)P2 pool dynamics detected by the coincidence biosensor tubbyCT maintain the integrity of ER-PM junctions during PLC signaling

Phosphoinositides (PIs) are important signaling molecules and determinants of membrane identity in the eukaryotic plasma membrane, where they multi-task in divergent signaling pathways. Signaling pleiotropy likely depends on distinct PI pools in the same membrane, although the physical definition of such pools has remained ambiguous. PI(4,5)P2, specifically, is also the precursor for the second messengers in the Gq/PLC pathway, IP3 and DAG, and is broken down by PLC{beta} during signaling. Endoplasmic reticulum-plasma membrane contact sites (ER-PM junctions) have emerged as central hubs for lipid transport between both membranes, and specifically for PI homeostasis by supplying the PM with phosphatidylinositol. Here we show that the tubby protein, by virtue of its C-terminal tubby-domain, preferentially localizes to ER-PM junctions by binding to both PI(4,5)P2 and the ER-PM tether E-Syt3. Under conditions of vigorous PI(4,5)P2 consumption by PLC{beta}, additional recruitment of tubby revealed an increase of a local PI(4,5)P2 pool fed by local synthesis through PI kinases. Inhibition of this pool-filling process led to the release of the ER-PM tethers, E-Syts, from the membrane and hence to loss of integrity of the ER-PM contact sites. We conclude that spatiotemporal metabolic channeling of PI synthesis initiated by non-vesicular transport in the ER-PM junctions specifies a local pool of PI(4,5)P2 that is pivotal for the maintenance of homeostatic functions during global depletion of PI(4,5)P2. The findings further suggest that the tubby-like proteins (TULPs), so far known to impact on energy homeostasis and obesity through primary cilia signaling, have an additional function at ER-PM junctions. HIGHLIGHTSO_LIThe tubby domain preferentially assembles into ER-PM junctions due to coincidence detection of PI(4,5)P2 and E-Syt3 C_LIO_LITubby recruitment reveals an increase of a local pool of PI(4,5)P2 in ER-PM junctions during PLC{beta} signaling C_LIO_LIJunctional PI(4,5)P2 dynamics require local synthesis of PI(4,5)P2 C_LIO_LILocal PI(4,5)P2 supply is required for integrity of ER-PM junctions during PLC{beta} activity. C_LI

cell biology↗

bioRxiv2020

A second PI(4,5)P2 binding site determines PI(4,5)P2 sensitivity of the tubby domain

Phosphosinositides (PIs) are lipid signaling molecules that operate by recruiting proteins to cellular membranes via PI recognition domains. Such domains are also used widely as fluorescence-coupled biosensors for cellular PIs. For PI(4,5)P2, the dominant PI of the plasma membrane (PM), only two recognition domains have been characterized in detail and used as sensors. One of them, the tubby domain, which is conserved in the tubby-like protein (TULP) family, is essential for targeting proteins into cilia in a process involving reversible membrane association. However, the PI(4,5)P2 binding properties of tubby domains have remained enigmatic. Here we used coarse-grained molecular dynamics (MD) simulations to explore PI(4,5)P2 binding by the prototypic tubby domain (tubbyCT). While the MD simulations showed a comparatively low PI(4,5)P2 affinity of the previously described canonical binding site, they unexpectedly revealed an adjacent second binding site, consisting of a conserved cationic cluster at the protein-membrane interface. Population of this second site dramatically increased membrane association of tubbyCT. Although less specific than the canonical binding pocket, this second site preferred binding of PI(4,5)P2 over PI(4)P and phosphatidyl serine. Mutations in this site impaired PI(4,5)P2-dependent PM localization in living cells and PI(4,5)P2 interaction in silico. Thus, the second binding site essentially contributes to the effective affinity and hence PM association of the tubby domain. The two-ligand binding mode may serve to sharpen the membrane association-dissociation cycle of TULPs that underlies delivery of ciliary cargo.

biophysics↗