Collagen deposition and alignment in the tumor stroma stiffen the extracellular matrix and, via integrin signaling, stimulate proton extrusion by cancer cells. In aggressive tumors, migration occurs preferentially along aligned collagen fibers, where proton efflux at the leading edge acidifies and degrades the matrix to facilitate invasion. Whether directional proton diffusion along collagen fibers exacerbates invasion remains uncertain, as it is not known how far protons can travel along a fiber before being released. To characterize the release barrier, we labeled collagen lysine side chains with a pH-sensitive fluorescein derivative and introduced an interfacial proton flux by microinjecting HCl near the surface of self-assembled collagen fibers elevated above the chamber bottom. Proton wave propagation was monitored by wide-field fluorescence microscopy. Quantitative analysis of the fluorescence transients revealed that proton migration is best described by a one-dimensional diffusion model with a finite surface-to-bulk release rate. A single global parameter set consistently described all experiments. From the fitted release rate, we determined a Gibbs activation energy barrier ΔG‡ of approximately 30 kBT, comparable to values reported for membrane–water interfaces. These results demonstrate that protons can travel tens of micrometers along collagen fibers before equilibrating with the bulk solution, suggesting that membrane-anchored collagen fibers may provide an effective pathway for directing acidification along migration tracks in tumor stroma.

We report a molecular strategy for precise, reversible, and noninvasive photoregulation of ion-selective membrane transport. Embedding azobenzene-containing photolipids into bilayers enables nanoscale control over the interaction and mobility of small-molecule ion carriers. Photoisomerization alone produces only minor changes in baseline conductance, consistent with the limited influence of small bilayer thickness variations on ion permeability, yet it elicits striking responses in the presence of mobile carriers. A newly designed protonophore exhibits proton-selective currents that increase by up to 200-fold under UV illumination and revert to baseline within milliseconds upon blue light. These effects cannot be explained by thickness or fluidity changes. Instead, they arise from light-dependent interactions between azobenzene moieties and the carrier that increase the membrane-bound carrier concentration and lower the effective barrier for transbilayer permeation via interfacial dipole and packing modulation. Because this mechanism relies entirely on chemical design - without genetic modification - and is compatible with photoswitches operating at longer wavelengths, it establishes a versatile framework for dynamic, light-driven control of ion transport in biological membranes and synthetic nanosystems.

Proton gradients power diverse biological processes, yet how interfacial proton migration is regulated remains unclear. Here we quantify how membrane composition controls interfacial proton migration using an approach that releases protons directly at the surface of a membrane patch via an embedded ionophore. Fluorometrically monitoring proton arrival at a distant patch across neutral, negatively charged, and positively charged membranes, we confirm that both the lateral surface diffusion coefficient and the activation barrier for proton release into the bulk vary rather modestly. In contrast to membrane electrostatics, membrane incorporation of glycolipids typical of thylakoid membranes—digalactosyldiacylglycerol and sulfoquinovosyldiacylglycerol—leads to a more pronounced reduction of the lateral proton diffusion coefficient, with comparatively small effects on the surface-to-bulk release barrier. Thus, interfacial proton migration is governed primarily by hydration-layer properties rather than membrane charge. These results establish membrane-anchored sugars as potent modulators of long-range proton conduction and provide a mechanistic framework for localized proton coupling in glycolipid-rich biological membranes.

The stability of membrane proteins depends on amino acid-specific interactions within the protein structure, its surroundings, e.g. the lipid membrane and ionic solutions, and their substrates. A molecular understanding of the factors affecting protein stability is essential for comprehending the respective structure–function relationships. This study aims to investigate the influence of native cysteine residues, ions, and glycerol on the stability of GlpF, the aqua(glycerol)porin of E. coli, under well-controlled conditions. To this end, the wild-type protein and its variant with four cysteine residues in the transmembrane helix bundle mutated to glycines were overexpressed and purified. The thermal unfolding of GlpF occurs at a transition temperature of 59.2 ± 1.4 °C, corresponding to an apparent Gibbs free energy of unfolding of approximately 10 kcal/mol. In contrast to the wild-type protein, the mutated GlpF exhibits a second unfolding transition, lowered by approximately 10 °C, and a decreased Gibbs free energy of unfolding of 5 kcal/mol. Our findings highlight that divalent ions and glycerol have a more pronounced stabilizing effect on the mutated GlpF than the wild-type protein. The structural consequences of cysteine to glycine mutations in terms of transmembrane helix rearrangement, the inter- and intra-chain H-bond network, and the reduced inter-chain interaction free energy, as well as intra-chain electrostatic energy and van der Waals energy, are revealed by molecular dynamics simulations. In conclusion, our findings illustrate the significance of cysteine residues distant from each other on protein oligomerization and stability, and the amplified impact of (de)stabilizing agents on less stable protein variants.

Voltage-gated potassium (Kv) channels, essential for shaping action potentials, are influenced not only by voltage but also by mechanical stimuli. Membrane tension has been shown to stabilize the open state of the pore, suggesting that pore dilation occurs in a thinner membrane. However, structural studies using cryo-electron microscopy indicate that membrane thinning is associated with pore closure. To clarify the role of local hydrophobic membrane thickness in channel gating, we reconstituted Kv channels from Aeropyrum pernix (KvAP) into planar bilayers containing photoswitchable lipids. Blue light exposure increased membrane thickness and increased the KvAP activity at both single channel and ensemble levels, whereas UV light reversed these effects by decreasing thickness and reducing activity. These results support a model in which the positioning of lipid phosphates in a thicker membrane drives the upward movement of interacting arginine residues in the KvAP voltage sensor and primes the channel for opening. This light-driven regulation of Kv channel activity offers new possibilities for controlling neuronal excitability. ### Competing Interest Statement The authors have declared no competing interest.

The human voltage-gated proton channel Hv1 has an intriguing architecture, as (i) its voltage-sensing unit contributes to the proton permeation pathway and (ii) the single channel proton flux is too large to be supplied solely by diffusion from the aqueous surroundings, and it likely involves both titratable residues and protein-bound water molecules. The paths followed by protons being transferred through the channel, and the identity of the protein groups that can transiently bind protons, remain unclear. To investigate the dynamic hydrogen-bond paths sampled at the channel mouths and inside the pore we carried out extensive atomic-level molecular dynamics simulations and graph-based analyses. We found that the inter-helical region of the channel hosts an extensive water-mediated hydrogen-bond network that includes the key aspartic residue essential for proton selectivity and additional titratable sidechains. We implemented a graph-based protocol to identify continuous hydrogen-bond paths that can connect the aspartic residue to either side of the membrane. We report that the internal H-bond network connects to lipid headgroups at both membrane interfaces, which could provide a mechanism for (i) the surrounding lipid membrane to influence the protein conformational dynamics and the internal hydrogen-bond network, and (ii) channeling rapidly diffusing protons from the membrane surface into and out of the Hv1 pore.

Voltage-gated potassium (Kv) channels are e ssential for shaping action potentials and rely on anionic lipids for proper gating, yet the mechanistic basis of lipid–channel interactions remains unclear. Cryo-electron microscopy studies suggest that, in the down state, arginine residues of the voltage sensor draw lipid phosphates upward, leading to a local membrane thinning of ~5 Å—an effect absent in the open state. To test whether membrane thickness directly affects voltage sensor function, we reconstituted Kv channels from Aeropyrum pernix (KvAP) into planar lipid bilayers containing photoswitchable lipids. Upon blue light illumination, the membrane thickened, and KvAP activity increased; UV light reversed both effects. Our findings indicate that membrane thickening weakens the interaction between lipid phosphates and voltage-sensing arginines in the down state, lowering the energy barrier for the transition to the up state and thereby promoting channel opening. This non-genetic, membrane-mediated approach provides a new strategy to control ion channel activity using light and establishes a direct, reversible link between membrane mechanics and voltage sensing, with potential applications in the remote control of neuronal excitability.

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 that are too low to prevent membrane-protein aggregation. Additionally, the identification of proteins is hampered by the lack of suitable molecular-weight markers such as 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 the 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), show only negligible aggregation, as evidenced by the fluorescent brightnesses and diffusion times of individual nanodiscs. In addition, 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.

The regulation of ion transport across biological membranes using light is a powerful research tool with potential therapeutic applications. Microbial channelrhodopsins, widely used in optogenetics, enable passive photocurrents that facilitate advanced studies of synaptic plasticity and neuronal connectivity. However, their applicability is limited by the need for genetic transfection to introduce channelrhodopsins into target cells. Here, we present a synthetic alternative combining small-molecule carriers with azobenzene-containing photolipids (OptoDArG) to achieve rapid and reversible ion-selective permeability modulation by light. Incorporating a novel lipidated nile blue derivative (NB-lipid) into photoswitchable bilayers enabled fully reversible ≈200-fold on–off modulation of H+ currents under UV and blue light illumination. The transport kinetics classify NB-lipids as cationic protonophores, demonstrating high sensitivity to OptoDArG-mediated changes in lipid packing and bilayer thickness. Another protonophore, carbonyl cyanide m -chlorophenylhydrazone (CCCP), exhibited similar sensitivity, though to a lesser extent. This concept extends beyond protonophores: valinomycin-mediated K+ currents showed several-fold increases under UV light, rapidly reversed by blue light-induced generation of OptoDArG's trans photoisomer. Our approach demonstrates that rapid, non-invasive, spatially precise, and reversible light-triggered currents can be achieved without genetic modifications. This strategy, exemplified by H+-selective currents, may be extended to other ions through tailored carrier design. ### Competing Interest Statement The authors have declared no competing interest.

Molecular interactions under steric confinement are important in chemistry, biology, biotechnology, and medicine. The impact of nanoconfinement on the underpinning kinetics and affinities is, however, unclear. While theoretical frameworks predict any effect only for very fast diffusion-limited association kinetics, experimental studies report that molecules can be trapped inside confined spaces to increase the effective local concentration and impact binding kinetics. Understanding is furthermore complicated by poorly comparable confinement geometries and reactions. Here, we determine the kinetics and affinities for interactions slower than the diffusion limit using highly modular DNA origami nanopores as model nanoconfinement systems. The pores feature either inside or outside their narrow lumen a single receptor, which can bind to three differently sized biomolecular ligands. We conduct kinetic binding analysis at the single-molecule resolution using fluorescence correlation spectroscopy to readily acquire large datasets and help overcome limitations of other single-molecule approaches. Nanoconfinement is found to hinder ligand association and dissociation, even below the diffusion limit. Yet, both suppressed kinetics compensate for each other to yield the same overall equillibrium affinity as nonconfined receptors, while local concentration enhancement by ligand trapping was not observed. We expect our insights and experimental strategy to guide the development of biosensing nanopores and help advance the understanding of biological nanochannels.

Fluorescent labeling of membrane proteins is essential for exploring their functions, signaling pathways, interaction partners, and structural dynamics. Organic fluorophores are commonly used for this purpose due to their favorable photophysical properties and photostability. However, a persistent challenge is the inaccessibility of the surface-exposed cysteine residues required for site-specific labeling, as these residues often become sequestered within detergent micelles during protein extraction. To address this limitation, we developed an approach based on polymer-encapsulated nanodiscs that preserves the protein’s native-like lipid-bilayer environment while ensuring the accessibility of surface-exposed cysteine residues. In this method, His-tagged proteins embedded in native nanodiscs are retained on a nickel affinity column, allowing for simultaneous purification and labeling by adding fluorescent dyes. This versatile technique was demonstrated with two challenging-to-label membrane proteins, the potassium channel KvAP and the urea channel HpUreI, for which detergent-based labeling had failed. This opens new possibilities for studying a wide range of fluorescently labeled membrane proteins in near-native states, advancing applications in biophysics, structural biology, and drug discovery.

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.

Light-triggered cell excitation or inhibition has manifold applications for research or therapeutic purposes. Most often, optogenetics or soft electronics have been used for this purpose. In a complementary approach, we exploited lipids with azobenzene moieties to achieve the same effect. Here, we show that part of the effect that photo-susceptible lipids exert on cellular excitability is due to their ability to alter mechanical membrane tension. To this end, we recorded the changes in the activity of purified and reconstituted potassium ion channels and tension simultaneously.

Proton translocation through lipid membranes is a fundamental process in the field of biology. Several theoretical models have been developed and presented over the years to explain the phenomenon, yet the exact mechanism is still not well understood. Here, we show that proton translocation is directly related to membrane potential fluctuations. Using high-throughput wide-field second harmonic (SH) microscopy, we report apparently universal transmembrane potential fluctuations in lipid membrane systems. Molecular simulations and free energy calculations suggest that H+ permeation proceeds predominantly across a thin, membrane-spanning water needle and that the transient transmembrane potential drives H+ ions across the water needle. This mechanism differs from the transport of other cations that require completely open pores for transport and follows naturally from the well-known Grotthuss mechanism for proton transport in bulk water. Furthermore, SH imaging and conductivity measurements reveal that the rate of proton transport depends on the structure of the hydrophobic core of bilayer membranes.