Dual mechanisms contribute to enhanced voltage dependence of an electric fish potassium channel.

The voltage dependence of different voltage-gated potassium channels, described by the voltage at which half of the channels are open (V1/2), varies over a range of 80 mV and is influenced by factors such as the number of positive gating charges and the identity of the hydrophobic amino acids in the channel's voltage sensor (S4). Here we explore by experimental manipulations and molecular dynamics simulation the contributions of two derived features of an electric fish potassium channel (Kv1.7a) that is among the most voltage-sensitive Shaker family potassium channels known. These are a patch of four contiguous negatively charged glutamates in the S3-S4 extracellular loop and a glutamate in the S3b helix. We find that these negative charges affect V1/2 by separate, complementary mechanisms. In the closed state, the S3-S4 linker negative patch reduces the membrane surface charge biasing the channel to enter the open state while, upon opening, the negative amino acid in the S3b helix faces the second (R2) gating charge of the voltage sensor electrostatically biasing the channel to remain in the open state. This work highlights two evolutionary novelties that illustrate the potential influence of negatively charged amino acids in extracellular loops and adjacent helices to voltage dependence.

Regulation and drug modulation of a voltage-gated sodium channel: Pivotal role of the S4-S5 linker in activation and slow inactivation.

Voltage-gated sodium (NaV) channels control excitable cell functions. While structural investigations have revealed conformation details of different functional states, the mechanisms of both activation and slow inactivation remain unclear. Here, we identify residue T140 in the S4-S5 linker of the bacterial voltage-gated sodium channel NaChBac as critical for channel activation and drug effects on inactivation. Mutations at T140 either attenuate activation or render the channel nonfunctional. Propofol, a clinical anesthetic known to inhibit NaChBac by promoting slow inactivation, binds to a pocket between the S4-S5 linker and S6 helix in a conformation-dependent manner. Using 19F-NMR to quantify site-specific binding by saturation transfer differences (STDs), we found strong STDs in inactivated, but not activated, NaChBac. Molecular dynamics simulations show a highly dynamic pocket in the activated conformation, limiting STD buildup. In contrast, drug binding to this pocket promotes and stabilizes the inactivated states. Our results provide direct experimental evidence showing distinctly different associations between the S4-S5 linker and S6 helix in activated and inactivated states. Specifically, an exchange occurs between interaction partners T140 and N234 of the same subunit in activation, and T140 and N225 of the domain-swapped subunit in slow inactivation. The drug action on slow inactivation of prokaryotic NaV channels seems to have a mechanism similar to the recently proposed "door-wedge" action of the isoleucine-phenylalanine-methionine (IFM) motif on the fast inactivation of eukaryotic NaV channels. Elucidating this gating mechanism points to a possible direction for conformation-dependent drug development.

Fluctuation Theorems for Heat Exchanges between Passive and Active Baths.

In addition to providing general constraints on probability distributions, fluctuation theorems allow us to infer essential information on the role played by temperature in heat exchange phenomena. In this numerical study, we measure the temperature of an out-of-equilibrium active bath using a fluctuation theorem that relates the fluctuations in the heat exchanged between two baths to their temperatures. Our setup consists of a single particle moving between two wells of a quartic potential accommodating two different baths. The heat exchanged between the two baths is monitored according to two definitions: as the kinetic energy carried by the particle whenever it jumps from one well to the other and as the work performed by the particle on one of the two baths when immersed in it. First, we consider two equilibrium baths at two different temperatures and verify that a fluctuation theorem featuring the baths temperatures holds for both heat definitions. Then, we introduce an additional Gaussian coloured noise in one of the baths, so as to make it effectively an active (out-of-equilibrium) bath. We find that a fluctuation theorem is still satisfied with both heat definitions. Interestingly, in this case the temperature obtained through the fluctuation theorem for the active bath corresponds to the kinetic temperature when considering the first heat definition, while it is larger with the second one. We interpret these results by looking at the particle jump phenomenology.

Nonequilibrium Thermodynamics of DNA Nanopore Unzipping.

Using theory and simulations, we carried out a first systematic characterization of DNA unzipping via nanopore translocation. Starting from partially unzipped states, we found three dynamical regimes depending on the applied force f: (i) heterogeneous DNA retraction and rezipping (f<17 pN), (ii) normal (17 pN60 pN) drift-diffusive behavior. We show that the normal drift-diffusion regime can be effectively modeled as a one-dimensional stochastic process in a tilted periodic potential. We use the theory of stochastic processes to recover the potential from nonequilibrium unzipping trajectories and show that it corresponds to the free-energy landscape for single-base-pair unzipping. Applying this general approach to other single-molecule systems with periodic potentials ought to yield detailed free-energy landscapes from out-of-equilibrium trajectories.

Work Fluctuations for a Harmonically Confined Active Ornstein-Uhlenbeck Particle.

We study the active work fluctuations of an active Ornstein-Uhlenbeck particle in the presence of a confining harmonic potential. We tackle the problem analytically both for stationary and generic uncorrelated initial states. Our results show that harmonic confinement can induce singularities in the active work rate function, with linear stretches at large positive and negative active work, at sufficiently large active and harmonic force constants. These singularities originate from big jumps in the displacement and in the active force, occurring at the initial or ending points of trajectories and marking the relevance of boundary terms in this problem.

Dynamics of Motility-Induced Clusters: Coarsening beyond Ostwald Ripening.

We study the dynamics of clusters of active Brownian disks generated by motility-induced phase separation, by applying an algorithm that we devised to track cluster trajectories. We identify an aggregation mechanism that goes beyond Ostwald ripening but also yields a dynamic exponent characterizing the cluster growth z=3, in the timescales explored numerically. Clusters of mass M self-propel with enhanced diffusivity D∼Pe^{2}/sqrt[M]. Their fast motion drives aggregation into large fractal structures, which are patchworks of diverse hexatic orders, and coexist with regular, orientationally uniform, smaller ones. To bring out the impact of activity, we perform a comparative study of a passive system that evidences major differences with the active case.

Novel Isoform DTX3c Associates with UBE2N-UBA1 and Cdc48/p97 as Part of the EphB4 Degradation Complex Regulated by the Autocrine IGF-II/IRA Signal in Malignant Mesothelioma.

EphB4 angiogenic kinase over-expression in Mesothelioma cells relies upon a degradation rescue signal provided by autocrine IGF-II activation of Insulin Receptor A. However, the identity of the molecular machinery involved in EphB4 rapid degradation upon IGF-II signal deprivation are unknown. Using targeted proteomics, protein-protein interaction methods, PCR cloning, and 3D modeling approaches, we identified a novel ubiquitin E3 ligase complex recruited by the EphB4 C tail upon autocrine IGF-II signal deprivation. We show this complex to contain a previously unknown N-Terminal isoform of Deltex3 E3-Ub ligase (referred as "DTX3c"), along with UBA1(E1) and UBE2N(E2) ubiquitin ligases and the ATPase/unfoldase Cdc48/p97. Upon autocrine IGF-II neutralization in cultured MSTO211H (a Malignant Mesothelioma cell line that is highly responsive to the EphB4 degradation rescue IGF-II signal), the inter-molecular interactions between these factors were enhanced and their association with the EphB4 C-tail increased consistently with the previously described EphB4 degradation pattern. The ATPase/unfoldase activity of Cdc48/p97 was required for EphB4 recruitment. As compared to the previously known isoforms DTX3a and DTX3b, a 3D modeling analysis of the DTX3c Nt domain showed a unique 3D folding supporting isoform-specific biological function(s). We shed light on the molecular machinery associated with autocrine IGF-II regulation of oncogenic EphB4 kinase expression in a previously characterized IGF-II+/EphB4+ Mesothelioma cell line. The study provides early evidence for DTX3 Ub-E3 ligase involvement beyond the Notch signaling pathway.

Environment-Dependent Stability and Mechanical Properties of DNA Origami Six-Helix Bundles with Different Crossover Spacings.

The internal design of DNA nanostructures defines how they behave in different environmental conditions, such as endonuclease-rich or low-Mg2+ solutions. Notably, the inter-helical crossovers that form the core of such DNA objects have a major impact on their mechanical properties and stability. Importantly, crossover design can be used to optimize DNA nanostructures for target applications, especially when developing them for biomedical environments. To elucidate this, two otherwise identical DNA origami designs are presented that have a different number of staple crossovers between neighboring helices, spaced at 42- and 21- basepair (bp) intervals, respectively. The behavior of these structures is then compared in various buffer conditions, as well as when they are exposed to enzymatic digestion by DNase I. The results show that an increased number of crossovers significantly improves the nuclease resistance of the DNA origami by making it less accessible to digestion enzymes but simultaneously lowers its stability under Mg2+ -free conditions by reducing the malleability of the structures. Therefore, these results represent an important step toward rational, application-specific DNA nanostructure design.

Global and local mechanical properties control endonuclease reactivity of a DNA origami nanostructure.

We used coarse-grained molecular dynamics simulations to characterize the global and local mechanical properties of a DNA origami triangle nanostructure. The structure presents two metastable conformations separated by a free energy barrier that is lowered upon omission of four specific DNA staples (defect). In contrast, only one stable conformation is present upon removing eight staples. The metastability is explained in terms of the intrinsic conformations of the three trapezoidal substructures. We computationally modeled the local accessibility to endonucleases, to predict the reactivity of twenty sites, and found good agreement with the experimental data. We showed that global fluctuations affect local reactivity: the removal of the DNA staples increased the computed accessibility to a restriction enzyme, at sites as distant as 40 nm, due to an increase in global fluctuation. These results raise the intriguing possibility of the rational engineering of allosterically modulated DNA origami.

Dynamics of supercoiled DNA with complex knots: large-scale rearrangements and persistent multi-strand interlocking.

Knots and supercoiling are both introduced in bacterial plasmids by catalytic processes involving DNA strand passages. While the effects on plasmid organization has been extensively studied for knotting and supercoiling taken separately, much less is known about their concurrent action. Here, we use molecular dynamics simulations and oxDNA, an accurate mesoscopic DNA model, to study the kinetic and metric changes introduced by complex (five-crossing) knots and supercoiling in 2 kbp-long DNA rings. We find several unexpected results. First, the conformational ensemble is dominated by two distinct states, differing in branchedness and knot size. Secondly, fluctuations between these states are as fast as the metric relaxation of unknotted rings. In spite of this, certain boundaries of knotted and plectonemically-wound regions can persist over much longer timescales. These pinned regions involve multiple strands that are interlocked by the cooperative action of topological and supercoiling constraints. Their long-lived character may be relevant for the simplifying action of topoisomerases.

Pore translocation of knotted DNA rings.

We use an accurate coarse-grained model for DNA and stochastic molecular dynamics simulations to study the pore translocation of 10-kbp-long DNA rings that are knotted. By monitoring various topological and physical observables we find that there is not one, as previously assumed, but rather two qualitatively different modes of knot translocation. For both modes the pore obstruction caused by knot passage has a brief duration and typically occurs at a late translocation stage. Both effects are well in agreement with experiments and can be rationalized with a transparent model based on the concurrent tensioning and sliding of the translocating knotted chains. We also observed that the duration of the pore obstruction event is more controlled by the knot translocation velocity than the knot size. These features should advance the interpretation and design of future experiments aimed at probing the spontaneous knotting of biopolymers.

TacoxDNA: A user-friendly web server for simulations of complex DNA structures, from single strands to origami.

Simulations of nucleic acids at different levels of structural details are increasingly used to complement and interpret experiments in different fields, from biophysics to medicine and materials science. However, the various structural models currently available for DNA and RNA and their accompanying suites of computational tools can be very rarely used in a synergistic fashion. The tacoxDNA webserver and standalone software package presented here are a step toward a long-sought interoperability of nucleic acids models. The webserver offers a simple interface for converting various common input formats of DNA structures and setting up molecular dynamics (MD) simulations. Users can, for instance, design DNA rings with different topologies, such as knots, with and without supercoiling, by simply providing an XYZ coordinate file of the DNA centre-line. More complex DNA geometries, as designable in the cadnano, CanDo and Tiamat tools, can also be converted to all-atom or oxDNA representations, which can then be used to run MD simulations. Though the latter are currently geared toward the native and LAMMPS oxDNA representations, the open-source package is designed to be further expandable. TacoxDNA is available at http://tacoxdna.sissa.it. © 2019 Wiley Periodicals, Inc.

Full Phase Diagram of Active Brownian Disks: From Melting to Motility-Induced Phase Separation.

We establish the complete phase diagram of self-propelled hard disks in two spatial dimensions from the analysis of the equation of state and the statistics of local order parameters. The equilibrium melting scenario is maintained at small activities, with coexistence between active liquid and hexatic order, followed by a proper hexatic phase, and a further transition to an active solid. As activity increases, the emergence of hexatic and solid order is shifted towards higher densities. Above a critical activity and for a certain range of packing fractions, the system undergoes motility-induced phase separation and demixes into low and high density phases; the latter can be either disordered (liquid) or ordered (hexatic or solid) depending on the activity.

Active dumbbells: Dynamics and morphology in the coexisting region.

With the help of molecular dynamics simulations we study an ensemble of active dumbbells in purely repulsive interaction. We derive the phase diagram in the density-activity plane and we characterise the various phases with liquid, hexatic and solid character. The analysis of the structural and dynamical properties, such as enstrophy, mean-square displacement, polarisation, and correlation functions, shows the continuous character of liquid and hexatic phases in the coexisting region when the activity is increased starting from the passive limit.

Phase Coexistence in Two-Dimensional Passive and Active Dumbbell Systems.

We demonstrate that there is a macroscopic coexistence between regions with hexatic order and regions in the liquid or gas phase over a finite interval of packing fractions in active dumbbell systems with repulsive power-law interactions in two dimensions. In the passive limit, this interval remains finite, similar to what has been found in two-dimensional systems of hard and soft disks. We did not find discontinuous behavior upon increasing activity from the passive limit.

The DTX Protein Family: An Emerging Set of E3 Ubiquitin Ligases in Cancer.

Until recently, Deltex (DTX) proteins have been considered putative E3 ligases, based on the presence of an E3 RING domain in their protein coding sequence. The human DTX family includes DTX1, DTX2, DTX3, DTX3L and DTX4. Despite the fact that our knowledge of this class of E3-ubiquitin ligases is still at an early stage, our understanding of their role in oncogenesis is beginning to unfold. In fact, recently published studies allow us to define specific biological scenarios and further consolidate evidence-based working hypotheses. According to the current evidence, all DTX family members are involved in the regulation of Notch signaling, suggesting a phylogenetically conserved role in the regulation of this pathway. Indeed, additional evidence reveals a wider involvement of these proteins in other signaling complexes and cancer-promoting mechanisms beyond NOTCH signaling. DTX3, in particular, had been known to express two isoform variants (DTX3a and DTX3b). The recent identification and cloning of a third isoform variant in cancer (DTX3c), and its specific involvement in EphB4 degradation in cancer cells, sheds further light on this group of proteins and their specific role in cancer. Herein, we review the cumulative knowledge of this family of E3 Ubiquitin ligases with a specific focus on the potential oncogenic role of DTX isoforms in light of the rapidly expanding findings regarding this protein family's cellular targets and regulated signaling pathways. Furthermore, using a comparative and bioinformatic approach, we here disclose a new putative motif of a member of this family which may help in understanding the biological and contextual differences between the members of these proteins.

Detecting Liquid-Liquid Phase Separations Using Molecular Dynamics Simulations and Spectral Clustering.

A stringent test of the accuracy of empirical force fields is reproducing the phase diagram of bulk phases and mixtures. Exploring the phase diagram of mixtures requires the detection of phase boundaries and critical points. In contrast to most solid-liquid transitions, in which a global order parameter (average density) can be used to discriminate between two phases, some demixing transitions entail relatively subtle changes in the local environment of each molecule. In such cases, finite sampling errors and finite-size effects make the identification of trends in local order parameters extremely challenging. Here we analyze one such example, namely a methanol/hexane mixture, and compute several local and global structural properties. We simulate the system at various temperatures and study the structural changes associated with demixing. We show that despite a seemingly continuous transformation between mixed and demixed states, the topological properties of the H-bond network change abruptly as the system crosses the demixing line. In particular, by using spectral clustering, we show that the distribution of cluster sizes develops a fat tail (as expected from percolation theory) in the vicinity of the critical point. We illustrate a simple criterion to identify this behavior, which results from the emergence of large system-spanning clusters from a collection of aggregates. We further tested the spectral clustering analysis on a Lennard-Jones system as a standard example of a system with no H-bonds, and also, in this case, we were able to detect the demixing transition.

Molecular states and spin crossover of hemin studied by DNA origami enabled single-molecule surface-enhanced Raman scattering.

The study of biologically relevant molecules and their interaction with external stimuli on a single molecular scale is of high importance due to the availability of distributed rather than averaged information. Surface enhanced Raman scattering (SERS) provides direct chemical information, but is rather challenging on the single molecule (SM) level, where it is often assumed to require a direct contact of analyte molecules with the metal surface. Here, we detect and investigate the molecular states of single hemin by SM-SERS. A DNA aptamer based G-quadruplex mediated recognition of hemin directs its placement in the SERS hot-spot of a DNA Origami Nanofork Antenna (DONA). The configuration of the DONA structure allows the molecule to be trapped at the plasmonic hot-spot preferentially in no-contact configuration with the metal surface. Owing to high field enhancement at the plasmonic hot spot, the detection of a single folded G-quadruplex becomes possible. For the first time, we present a systematic study by SM-SERS where most hemin molecule adopt a high spin and oxidation state (III) that showed state crossover to low spin upon strong-field-ligand binding. The present study therefore, provides a platform for studying biologically relevant molecules and their properties at SM sensitivity along with demonstrating a conceptual advancement towards successful monitoring of single molecular chemical interaction using DNA aptamers.

RNA Pore Translocation with Static and Periodic Forces: Effect of Secondary and Tertiary Elements on Process Activation and Duration.

We use MD simulations to study the pore translocation properties of a pseudoknotted viral RNA. We consider the 71-nucleotide-long xrRNA from the Zika virus and establish how it responds when driven through a narrow pore by static or periodic forces applied to either of the two termini. Unlike the case of fluctuating homopolymers, the onset of translocation is significantly delayed with respect to the application of static driving forces. Because of the peculiar xrRNA architecture, activation times can differ by orders of magnitude at the two ends. Instead, translocation duration is much smaller than activation times and occurs on time scales comparable at the two ends. Periodic forces amplify significantly the differences at the two ends, for both activation times and translocation duration. Finally, we use a waiting-times analysis to examine the systematic slowing downs in xrRNA translocations and associate them to the hindrance of specific secondary and tertiary elements of xrRNA. The findings provide a useful reference to interpret and design future theoretical and experimental studies of RNA translocation.

Dataset of numerical simulations for aeroelastic control of an aero engine compressor cascade using plasma actuators.

The dataset presented here regard the analysis reported in the research article entitled "Comparison of different plasma actuation strategies for aeroelastic control on a linear compressor cascade" De Giorgi et al. (2021) [1]. These data are related to the Computational Fluid Dynamics (CFD) assessment of different plasma actuation strategies for the aeroelastic control of an aero engine compressor cascade in subsonic flow conditions. The authors evaluated the accuracy of numerical computations using experimental results. Here, both experimental and raw data of the CFD simulations are presented.