Biology subjects

Pohl, P.

Publications and source records attributed to Pohl, P..

9 recordsLinked to original sources

bioRxiv2026

Optical control of membrane mechanics via global and red-light-catalyzed, leaflet-selective photolipid switching

Azobenzene photolipids are versatile actuators of membrane mechanics and protein function, yet long-wavelength control and leaflet-selective perturbations remain difficult to impose. Here, we address both limitations. First, we show that red-light excitation of a lipidated Nile Blue derivative (NB-lipid) catalyzes rapid and reversible cis[->]trans photoisomerization of co-localized azobenzene photolipids without requiring covalent conjugation. Second, we demonstrate that photocatalysis is predominantly intraleaflet, enabling leaflet-selective actuation by incorporating the membrane-targeted chromophore into only one bilayer leaflet. This enables on-demand and rapid writing of transbilayer mechanical asymmetry, which we track via lock-in capacitance signatures. Critically, symmetric and asymmetric leaflet mechanical perturbations can be evoked within the same membrane by illuminating with blue or red light, respectively. Photocatalysis of electron-rich azobenzenes is anticipated to proceed via the singlet excited state of NB-lipid and thus avoids triplet-state phototoxicity. We underscore the biocompatibility of catalysis by demonstrating the effect of leaflet-specific catalyzed perturbations on the ROS-sensitive mechanosensitive peptide ion channel gramicidin A. Our results establish a novel tool for dissecting the role of dynamic leaflet-specific mechanical changes in membrane remodeling and protein function.

biophysics↗

bioRxiv2025

Clear Native Gel Electrophoresis for the Purification of Fluorescently Labeled Membrane Proteins in Native Nanodiscs

Native gel electrophoresis techniques, such as blue or clear native gel electrophoresis (BNE or CNE), are widely used to separate and characterize proteins. However, in high-resolution CNE, mild anionic or neutral detergents are often used at concentrations too low to prevent membrane-protein aggregation. Additionally, the identification of proteins is hampered by the lack of suitable molecular-weight markers, like those used in SDS-PAGE. Here, we introduce a novel approach that combines charged polymer-encapsulated nanodiscs and fluorescence correlation spectroscopy (FCS) to address both challenges. Membrane proteins are first extracted using Glyco-DIBMA, a negatively charged amphiphilic copolymer. This enables the spontaneous formation of nanodiscs harboring the fluorescently labeled target protein within a native-like lipid-bilayer environment, which is confirmed by FCS. The nanodiscs are then subjected to detergent-free CNE. As the number of protomers increases, the nanodiscs grow larger resulting in increased migration distances in CNE due to higher charge densities. Crucially, the nanodiscs remain intact throughout CNE, as demonstrated by FCS analysis of resolubilized bands excised from the gels. Moreover, the membrane proteins used in this study: a potassium channel (KvAP), a sodium channel (NavMs), a water channel (GlpF), and a urea channel (HpUreI) do not aggregate, as shown by the fluorescent brightnesses and diffusion times of individual nanodiscs. Moreover, the oligomeric states of membrane proteins can be deduced from the brightness per nanodisc. Since purified membrane proteins remain within a native-like lipid-bilayer environment and avoid detergent exposure, they are immediately suitable for downstream structural and functional studies.

biophysics↗

bioRxiv2025

Photomodulation of Kv Channel Activity

Voltage-gated potassium (Kv) channels shape action potentials, with their activity dependent on anionic lipids and modulated by membrane tension. However, the mechanism by which lipids regulate channel gating is not entirely clear. Cryo-electron microscopy studies suggest that in the down state-- typically associated with the inactive channel--arginines of the voltage sensor interact with lipid phosphates, pulling them upward and locally thinning the membrane by [~]5 [A]. This thinning is absent in the up state of the sensor, which is typically associated with the active channel. To test whether membrane thickness influences gating, we reconstituted Aeropyrum pernix Kv (KvAP) channels into planar lipid bilayers containing photoswitchable lipids. Blue light increased membrane thickness and KvAP activity, while UV light reversed these effects. Our results indicate that membrane thickening disrupts voltage sensor-lipid phosphate interactions, lowering the activation barrier for the up-movement of the voltage sensor and thus, channel opening. The resulting leftward shift in the current-voltage relationship demonstrates a light-driven approach to modulating Kv channel activity, with potential applications in neuronal excitability control. SignificanceVoltage-gated Kv channels respond to membrane voltage in a manner influenced by lipid composition and membrane tension. In the inactive state, voltage-sensing arginines interact with lipid phosphates, and when these interactions are strained--such as by membrane thickening or possibly by membrane tension--less voltage is required for channel opening. We demonstrate this interdependence by showing that a light-triggered increase in membrane thickness reversibly enhances Kv channel activity, directly linking membrane mechanics to voltage sensing. This work also establishes a light-based strategy for spatially precise Kv channel modulation.

biophysics↗

bioRxiv2025

Phototriggered proton-selective carrier currents through photoswitchable membranes

Controlling the flux of specific ions across lipid bilayers is a central challenge for cellular life. Here, we present a general strategy for the precise, noninvasive, and reversible photoregulation of specific ion transport across lipid bilayer membranes: using embedded azobenzene-containing photolipids as dopants that effectively photo-control the membrane permeation of specific ion carriers. Photolipid (OptoDArG) embedding and photoisomerization causes only minor changes in background ion conductance, consistent with classical models of membrane transport wherein small changes in bilayer thickness have little effect on ion permeability. Yet, in the presence of ion carriers, photoisomerization instead results in order-of-magnitude jumps in ion flux. These jumps are observed with both cationic and anionic carriers; particular protonophores deliver exceptional performance, with proton currents amplified by up to {approx}200-fold upon UV illumination yet reversed by blue light exposure. A model in which interfacial uptake and release reactions are modulated by light-induced membrane changes explains these results. Doping photolipids together with well-established selective ion carriers into membranes thus presents a flexible and general chemical strategy for reversible, millisecond-scale photoregulation of specific ion trans-port across lipid bilayers. It achieves large currents, long-lasting effects, its ion scope is easily tuned by carrier choice, no genetic modifications are needed, and it has the potential to harness recent photoswitch advances to rationally shift its single-photon action spectra from the UV/Vis right up to the NIR/SWIR: a convenient all-chemical approach toward light-based ion permeability regulation in the biological or synthetic cell. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=143 SRC="FIGDIR/small/632814v2_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@ed17aborg.highwire.dtl.DTLVardef@1ace648org.highwire.dtl.DTLVardef@42aef6org.highwire.dtl.DTLVardef@1eccced_HPS_FORMAT_FIGEXP M_FIG Graphical abstract C_FIG

biophysics↗

bioRxiv2023

Photolipid excitation triggers depolarizing optocapacitive currents and action potentials

Optically-induced changes in membrane capacitance may regulate neuronal activity without requiring genetic modifications. Previously, they mainly relied on sudden temperature jumps due to light absorption by membrane-associated nanomaterials or water. Yet, nanomaterial targeting or the required high infrared light intensities obstruct broad applicability. Now, we propose a very versatile approach: photolipids (azobenzene-containing diacylglycerols) mediate light-triggered cellular de- or hyperpolarization. As planar bilayer experiments show, the respective currents emerge from millisecond-timescale changes in bilayer capacitance. UV light changes photolipid conformation, which awards embedding plasma membranes with increased capacitance and evokes depolarizing currents. They open voltage-gated sodium channels in cells, generating action potentials. Blue light reduces the area per photolipid, decreasing membrane capacitance and eliciting hyperpolarization. If present, mechanosensitive channels respond to the increased mechanical membrane tension, generating large depolarizing currents that elicit action potentials. Membrane self-insertion of administered photolipids and focused illumination allows cell excitation with high spatiotemporal control. HighlightsO_LIRapid photolipid photoisomerization generates optocapacitive currents in planar lipid bilayers and HEK293 cells. C_LIO_LIThese currents originate from photo-induced changes in membrane capacitance C_LIO_LIUV light-triggered membrane depolarization opens NaV1.3, evoking action potentials. C_LIO_LIBlue light-induced mechanosensitive channel opening gives rise to depolarizing currents, which may evoke NaV1.3-mediated action potentials. C_LI

biophysics↗

bioRxiv2023

YidC from Escherichia coli forms an ion-conducting pore upon activation by ribosomes.

The universally conserved protein YidC aids the insertion and folding of transmembrane polypeptides independently or as a part of the SecYEG translocon complex. In the former scenario, the lipid-exposed YidC surface equipped with a highly conserved positively charged arginine is thought to facilitate membrane insertion of the nascent chain by providing a countercharge for the acidic residues at the polypeptides N-terminal region. Here we show that the purified and reconstituted E. coli YidC forms an ion-conducting transmembrane pore upon binding a ribosome or ribosome-nascent chain complex (RNC). This pore is closed in the absence of ribosomes. As this pore is not visible in the published monomeric YidC structure, we used AlphaFold to construct the model of a parallel YidC dimer. Experimental evidence for a dimeric assembly comes from our BN-PAGE analysis of native vesicles, fluorescence correlation spectroscopy studies, and single-molecule fluorescence microscopy. In the dimeric model, the conserved positively charged arginine and many residues interacting with nascent chains point into the putative pore. This result suggests the possibility of an alternative mode of YidC-assisted membrane protein insertion.

biophysics↗

bioRxiv2023

Steady-state polypeptide transfer from the translocon to the membrane

In concert with irreversible non-equilibrium peptide translation by the ribosome, the nascent polypeptide chain may integrate into the membrane or translocate to the other side of the membrane, facilitated by the conserved protein translocation channel SecYEG in bacteria and Sec61 in eukaryotes. Assuming equilibrium for the decision processes yielded the biological hydrophobicity scale, reflecting free-energy differences {Delta}G between the pore interior and membrane. Yet kinetic effects and molecular dynamic simulations suggested that a nascent polypeptide could not sample the two separate environments a sufficient number of times for partitioning in equilibrium. Here we tested the hypothesis employing purified and reconstituted SecYEG harboring a stalled ribosome nascent chain (RNC). The SecYEG-RNC complex was open in a de-energized membrane, allowing ion flow. Application of a membrane potential closed the channel if nascent chain hydrophobicity permitted membrane integration. Taking the ratio of steady-state to initial ion conductances as a measure of nascent chain hydrophobicity, we found {Delta}G for KvAPs voltage sensor (4th helix harboring four arginines) and FtsQs transmembrane helix to be equal to 0.3 and -2.1 kcal/mol, respectively. Thus, {Delta}G observed in our minimalistic system agrees very well with the position-dependent amino acid contribution of the biological hydrophobicity scale. Characteristic sampling times of ~2 s appear sufficient to reach a steady state for a ~20 amino acid-long segment invalidating the hypothesis of insufficient sampling.

biophysics↗

bioRxiv2022

Trapped pore waters in the open proton channel HV1

The voltage-gated proton channel, HV1, is crucial for innate immune responses. According to alternative hypotheses, protons either hop on top of an uninterrupted water wire or bypass titratable amino acids, interrupting the water wire halfway across the membrane. To distinguish between both hypotheses, we estimate the water mobility for the putative case of an uninterrupted wire. The predicted single-channel water permeability 3x10-12cm3s-1 reflects the permeability-governing number of hydrogen bonds between water molecules in single-file configuration and pore residues. However, the measured unitary water permeability does not confirm the prediction, i.e., it is negligible. Osmotic deflation of reconstituted lipid vesicles reveals trapped water inside the HV1 wild-type channel and D174A mutant open at 0 mV. The conductance of 1400 H+ s-1 per wild-type channel agrees with the calculated diffusion limit for a ~2 [A] capture radius for protons. Removal of a charged amino acid (D174) at the pore mouth decreases H+ conductance, conceivably by reducing the capture radius. At least one intervening amino acid contributes to H+ conductance while blocking water flow.

biophysics↗

bioRxiv2019

Binding of the motor protein SecA to the bacterial protein translocation channel SecYEG in the absence of ATP

Translocation of many secretory proteins through the bacterial plasma membrane is facilitated by a complex of the SecYEG channel with the motor protein SecA. The ATP-free complex is unstable in detergent, raising the question how SecA may perform several rounds of ATP hydrolysis without being released from the membrane embedded SecYEG. Here we show that dual recognition of (i) SecYEG and (ii) vicinal acidic lipids confers an apparent nanomolar affinity. High-speed atomic force microscopy visualizes the complexes between monomeric SecA and SecYEG as being stable for tens of seconds. These long-lasting events and complementary shorter ones both give rise to single ion channel openings of equal duration. Furthermore, luminescence resonance energy transfer reveals two conformations of the SecYEG-SecA complex that differ in the protrusion depth of SecAs two-helix finger into SecYEGs aqueous channel. Such movement of the finger is in line with the power stroke mechanism of protein translocation.

biophysics↗