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Antibiotics have been developed to effectively target and eliminate bacteria, but the rise in antimicrobial resistance (AR) complicates the treatment of certain infections. To address this issue, researchers have explored antimicrobial peptides (AMPs) that disrupt bacterial membranes. A promising method for this exploration is motif-based analysis, which identifies hidden patterns in AMPs to better understand their mechanism of action. While existing methods rely on expert knowledge, incorporating topic models can enhance analysis by revealing the contextual relationships between sequence elements. This is complemented by a data analytics tool designed to analyze AMP motifs and their biochemical properties. Such integration allows for the extraction of valuable motifs and the development of a robust data analytics module for predicting membrane activity. Additionally, we evaluated the biological relevance of motifs by extracting biochemical features, making structural predictions via Evolutionary Scale Modeling (ESM). Our results indicate that topic model-derived motifs are strongly associated with antimicrobial activity and demonstrate lower minimum inhibitory concentration values and capture contextual information more effectively than traditional frequency-based motifs. We also performed a comparative analysis between the two approaches regarding motif evolution, sequence-level attributes, and entropy measures, ultimately contributing to ongoing efforts to combat AR.

Herpesviruses are large viruses that infect nearly all vertebrates and some invertebrates and cause lifelong infections in most of the world’s population. During replication, herpesviruses export their capsids from the nucleus into the cytoplasm by an unusual mechanism in which the viral nuclear egress complex (NEC) deforms the nuclear membrane around the capsid. During replication of herpesviruses, capsids escape from the nucleus into the cytoplasm by budding at the inner nuclear membrane. This unusual process is mediated by the viral nuclear egress complex (NEC) that deforms the membrane around the capsid by oligomerizing into a hexagonal, membrane-bound scaffold. Here, we found that highly basic membrane-proximal regions (MPRs) of the NEC alter lipid order by inserting into the lipid headgroups and promote negative Gaussian curvature. We also find that the electrostatic interactions between the MPRs and the membranes are essential for membrane deformation. One of the MPRs is phosphorylated by a viral kinase during infection, and the corresponding phosphomimicking mutations block capsid nuclear egress. We show that the same phosphomimicking mutations disrupt the NEC-membrane interactions and inhibit NEC-mediated budding in vitro , providing a biophysical explanation for the in vivo phenomenon. Our data suggest that the NEC generates negative membrane curvature by both lipid ordering and protein scaffolding and that phosphorylation acts as an off switch that inhibits the membrane-budding activity of the NEC to prevent capsid-less budding. IMPORTANCE Herpesviruses are large viruses that infect nearly all vertebrates and some invertebrates and cause lifelong infections in most of the world’s population. During replication, herpesviruses export their capsids from the nucleus into the cytoplasm by an unusual mechanism in which the viral nuclear egress complex (NEC) deforms the nuclear membrane around the capsid. However, how membrane deformation is achieved is unclear. Here, we show that the NEC from herpes simplex virus 1, a prototypical herpesvirus, uses clusters of positive charges to bind membranes and order membrane lipids. Reducing the positive charge or introducing negative charges weakens the membrane deforming ability of the NEC. We propose that the virus employs electrostatics to deform nuclear membrane around the capsid and can control this process by changing the NEC charge through phosphorylation. Blocking NEC-membrane interactions could be exploited as a therapeutic strategy.

Hysteresis in the conductance of voltage-sensitive ion channels is observed when the transmembrane voltage is periodically varied with time. Although this phenomenon has been used in studies of gating of the voltage-dependent anion channel, VDAC, from the outer mitochondrial membrane for nearly four decades, full hysteresis curves have never been reported, because the focus was solely on the channel opening branches of the hysteresis loops. We studied the hysteretic response of a multichannel VDAC system to a triangular voltage ramp the frequency of which was varied over three orders of magnitude, from 0.5 mHz to 0.2 Hz. We found that in this wide frequency range the area encircled by the hysteresis curves changes by less than a factor of three, suggesting broad distribution of the characteristic times and strongly non-equilibrium behavior. At the same time, quasi-equilibrium two-state behavior is observed for hysteresis branches corresponding to VDAC opening. This enables calculation of the usual equilibrium gating parameters, gating charge and voltage of equipartitioning, which were found to be almost insensitive to the ramp frequency. To rationalize this peculiarity, we hypothesize that during voltage-induced closure and opening the system explores different regions of the complex free energy landscape, and, in the opening branch, follows quasi-equilibrium paths.

Nanopore-based sensing of charged biopolymers is a powerful single-molecule method. In aconventional nanopore experiment, a single biological (proteinaceous) or solid-state nanopore perforates a thin membrane that is wetted by, and electrically isolates, two opposing reservoirs of electrolyte solution. A potential is applied across the membrane via external electronics coupled to the electrolyte reservoirs with electrochemical electrodes, actuating the system. The electric field set up by the applied potential in the nanopore and its immediate environment plays two roles: supporting an ionic current through the nanopore, which reports on the properties of the pore and its contents; and acting on analyte molecules to attract them to, and drive them into, the nanopore. The presence of a large biopolymer in the pore modulates the ionic current ( ). The duration of the ionic current modulation corresponds to the length of time the polymer spends in the pore from capture to its ultimate escape, either by retraction to the reservoir from which it was captured, or by translocation to the opposite reservoir . The probabilities of retraction or translocation, or splitting probabilities, and the corresponding distributions of escape times ( esc ), are particularly sensitive to the size and charge of the analyte molecule and have been the focus of much theoretical, computational, and experimental effort. An underlying physical framework in which the distribution of escape times is modeled as a first-passage time from a one-dimensional potential is quantitatively predictive for a wide range of experiments. The complexity of this potential for the general case, however, requires calculations to guide experimental design that can be tedious to implement. PPDiffuse is intended to remove this burden from the nanopore research community and enable convenient, rational design of nanopore experiments with complex substrates such as polypeptides.

The voltage-dependent anion channel (VDAC) is the primary regulating pathway of water-soluble metabolites and ions across the mitochondrial outer membrane. When reconstituted into lipid membranes, VDAC responds to sufficiently large transmembrane potentials by transitioning to gated states in which ATP/ADP flux is reduced and calcium flux is increased. Two otherwise unrelated cytosolic proteins, tubulin, and α-synuclein (αSyn), dock with VDAC by a novel mechanism in which the transmembrane potential draws their disordered, polyanionic C-terminal domains into and through the VDAC channel, thus physically blocking the pore. For both tubulin and αSyn, the blocked state is observed at much lower transmembrane potentials than VDAC gated states, such that in the presence of these cytosolic docking proteins, VDAC’s sensitivity to transmembrane potential is dramatically increased. Remarkably, the features of the VDAC gated states relevant for bioenergetics—reduced metabolite flux and increased calcium flux—are preserved in the blocked state induced by either docking protein. The ability of tubulin and αSyn to modulate mitochondrial potential and ATP production in vivo is now supported by many studies. The common physical origin of the interactions of both tubulin and αSyn with VDAC leads to a general model of a VDAC inhibitor, facilitates predictions of the effect of post-translational modifications of known inhibitors, and points the way toward the development of novel therapeutics targeting VDAC.

Involvement of alpha-synuclein (αSyn) in Parkinson’s disease (PD) is complicated and difficult to trace on cellular and molecular levels. Recently, we established that αSyn can regulate mitochondrial function by voltage-activated complexation with the voltage-dependent anion channel (VDAC) on the mitochondrial outer membrane. When complexed with αSyn, the VDAC pore is partially blocked, reducing the transport of ATP/ADP and other metabolites. Further, αSyn can translocate into the mitochondria through VDAC, where it interferes with mitochondrial respiration. Recruitment of αSyn to the VDAC-containing lipid membrane appears to be a crucial prerequisite for both the blockage and translocation processes. Here we report an inhibitory effect of HK2p, a small membrane-binding peptide from the mitochondria-targeting N-terminus of hexokinase 2, on αSyn membrane binding, and hence on αSyn complex formation with VDAC and translocation through it. In electrophysiology experiments, the addition of HK2p at micromolar concentrations to the same side of the membrane as αSyn results in a dramatic reduction of the frequency of blockage events in a concentration-dependent manner, reporting on complexation inhibition. Using two complementary methods of measuring protein-membrane binding, bilayer overtone analysis and fluorescence correlation spectroscopy, we found that HK2p induces detachment of αSyn from lipid membranes. Experiments with HeLa cells using proximity ligation assay confirmed that HK2p impedes αSyn entry into mitochondria. Our results demonstrate that it is possible to regulate αSyn–VDAC complexation by a rationally designed peptide, thus suggesting new avenues in the search for peptide therapeutics to alleviate αSyn mitochondrial toxicity in PD and other synucleinopathies.

Dimeric tubulin, an abundant water-soluble cytosolic protein known primarily for its role in the cytoskeleton, is routinely found to be associated with mitochondrial outer membranes, although the structure and physiological role of mitochondria-bound tubulin are still unknown. There is also no consensus on whether tubulin is a peripheral membrane protein or is integrated into the outer mitochondrial membrane. Here the results of five independent techniques—surface plasmon resonance, electrochemical impedance spectroscopy, bilayer overtone analysis, neutron reflectometry, and molecular dynamics simulations—suggest that α-tubulin’s amphipathic helix H10 is responsible for peripheral binding of dimeric tubulin to biomimetic “mitochondrial” membranes in a manner that differentiates between the two primary lipid headgroups found in mitochondrial membranes, phosphatidylethanolamine and phosphatidylcholine. The identification of the tubulin dimer orientation and membrane-binding domain represents an essential step toward our understanding of the complex mechanisms by which tubulin interacts with integral proteins of the mitochondrial outer membrane and is important for the structure-inspired design of tubulin-targeting agents.

It is well established that α-synuclein (α-syn) binding from solution to the surface of membranes composed of negatively charged and/or non-lamellar lipids can be characterized by equilibrium dissociation constants of tens of micromolar. Previously, we have found that a naturally occurring nanopore of the mitochondrial voltage-dependent anion channel (VDAC), reconstituted into planar bilayers of a plant-derived lipid, responds to α-syn at nanomolar solution concentrations. Here, using lipid mixtures that mimic the composition of mitochondrial outer membranes, we show that functionally important binding does indeed take place in the nanomolar range. We demonstrate that the voltage-dependent rate at which a membrane-embedded VDAC nanopore captures α-syn is a strong function of membrane composition. Comparison of the nanopore results with those obtained by the bilayer overtone analysis of membrane binding demonstrates a pronounced correlation between the two datasets. The stronger the binding, the larger the on-rate, but with some notable exceptions. This leads to a tentative model of α-syn-membrane interactions, which assigns different lipid-dependent roles to the N- and C-terminal domains of α-syn accounting for both electrostatic and hydrophobic effects. As a result, the rate of α-syn capture by the nanopore is not simply proportional to the α-syn concentration on the membrane surface but found to be sensitive to the specific interactions of each domain with the membrane and nanopore.

The burgeoning field of nanotechnology aims to create and deploy nanoscale structures, devices, and systems with novel, size-dependent properties and functions. The nanotechnology revolution has sparked radically new technologies and strategies across all scientific disciplines, with nanotechnology now applied to virtually every area of research and development in the US and globally. NanoFlorida was founded to create a forum for scientific exchange, promote networking among nanoscientists, encourage collaborative research efforts across institutions, forge strong industry-academia partnerships in nanoscience, and showcase the contributions of students and trainees in nanotechnology fields. The 2019 NanoFlorida International Conference expanded this vision to emphasize national and international participation, with a focus on advances made in translating nanotechnology. This review highlights notable research in the areas of engineering especially in optics, photonics and plasmonics and electronics; biomedical devices, nano-biotechnology, nanotherapeutics including both experimental nanotherapies and nanovaccines; nano-diagnostics and -theranostics; nano-enabled drug discovery platforms; tissue engineering, bioprinting, and environmental nanotechnology, as well as challenges and directions for future research.