Search

Membrane lipids control the cellular activity of kinases containing the Src homology 2 (SH2) domain through direct lipid-SH2 domain interactions. Here we report development of novel non-lipidic small molecule inhibitors of the lipid-SH2 domain interaction that block the cellular activity of their host proteins. As a pilot study, we evaluated the efficacy of lipid-SH2 domain interaction inhibitors for spleen tyrosine kinase (Syk), which is implicated in hematopoietic malignancies, including acute myeloid leukemia (AML). An optimized inhibitor (WC36) specifically and potently suppressed oncogenic activities of Syk in AML cell lines and patient-derived AML cells. Unlike ATP-competitive Syk inhibitors, WC36 was refractory to de novo and acquired drug resistance due to its ability to block not only the Syk kinase activity but also its non-catalytic scaffolding function that is linked to drug resistance. Collectively, our study shows that targeting lipid-protein interaction is a powerful approach to developing novel small molecule drugs.

Phospholipid extrusion by ABC subfamily A (ABCA) exporters is central to cellular physiology, although the specifics of the underlying substrate interactions and transport mechanisms remain poorly resolved at the molecular level. Here we report cryo-EM structures of lipid-embedded human ABCA7 in an open state and in a nucleotide-bound, closed state at resolutions between 3.6 and 4.0 Å. The former reveals an ordered patch of bilayer lipids traversing the transmembrane domain (TMD), while the latter reveals a lipid-free, closed TMD with a small extracellular opening. These structures offer a structural framework for both substrate entry and exit from the ABCA7 TMD and highlight conserved rigid-body motions that underlie the associated conformational transitions. Combined with functional analysis and molecular dynamics (MD) simulations, our data also shed light on lipid partitioning into the ABCA7 TMD and localized membrane perturbations that underlie ABCA7 function and have broader implications for other ABCA family transporters.

Glutamate is the most abundant excitatory neurotransmitter in the central nervous system, and its precise control is vital to maintain normal brain function and to prevent excitotoxicity1. The removal of extracellular glutamate is achieved by plasma-membrane-bound transporters, which couple glutamate transport to sodium, potassium and pH gradients using an elevator mechanism2–5. Glutamate transporters also conduct chloride ions by means of a channel-like process that is thermodynamically uncoupled from transport6–8. However, the molecular mechanisms that enable these dual-function transporters to carry out two seemingly contradictory roles are unknown. Here we report the cryo-electron microscopy structure of a glutamate transporter homologue in an open-channel state, which reveals an aqueous cavity that is formed during the glutamate transport cycle. The functional properties of this cavity, combined with molecular dynamics simulations, reveal it to be an aqueous-accessible chloride permeation pathway that is gated by two hydrophobic regions and is conserved across mammalian and archaeal glutamate transporters. Our findings provide insight into the mechanism by which glutamate transporters support their dual function, and add information that will assist in mapping the complete transport cycle shared by the solute carrier 1A transporter family. Glutamate transporters conduct chloride ions through an aqueous channel with hydrophobic gates that forms during the glutamate transport cycle.

P-glycoprotein (Pgp) is a major efflux pump in humans, overexpressed in a variety of cancers and associated with the development of multi-drug resistance. Allosteric modulation by various ligands (e.g., transport substrates, inhibitors, and ATP) has been biochemically shown to directly influence structural dynamics, and thereby, the function of Pgp. However, the molecular details of such effects, particularly with respect to the role and involvement of the surrounding lipids, are not well established. Here, we employ all-atom molecular dynamics (MD) simulations to study the conformational landscape of Pgp in the presence of a high-affinity, third-generation inhibitor, tariquidar, in comparison to the nucleotide-free (APO) and the ATP-bound states, in order to characterize the mechanical effects of the inhibitor that might be of relevance to its blocking mechanism of Pgp. Simulations in a multi-component lipid bilayer show a dynamic equilibrium between open(er) and more closed inward-facing (IF) conformations in the APO state, with binding of ATP shifting the equilibrium towards conformations more prone to ATP hydrolysis and subsequent events in the transport cycle. In the presence of the inhibitor bound to the drug-binding pocket within the transmembrane domain (TMD), Pgp samples more open IF conformations, and the nucleotide binding domains (NBDs) become highly dynamic. Interestingly, and reproduced in multiple independent simulations, the inhibitor is observed to facilitate recruitment of lipid molecules into the Pgp lumen through the two proposed drug-entry portals, where the lipid head groups from the cytoplasmic leaflet penetrate into and, in some cases, translocate inside the TMD, while the lipid tails remain extended into the bulk lipid environment. These “wedge” lipids likely enhance the inhibitor-induced conformational restriction of the TMD leading to the differential modulation of coupling pathways observed with the NBDs downstream. We suggest a novel inhibitory mechanism for tariquidar, and potentially for related third-generation Pgp inhibitors, where lipids are seen to enhance the inhibitory role in the catalytic cycle of membrane transporters., Lipid invasion of P-glycoprotein, enhanced by binding of an inhibitor.

This work aims to address the effectiveness and challenges of using active infrared thermography (IRT) onboard an unmanned aerial vehicle (UAV) platform. The work seeks to assess the performance of small low-powered forms of excitation which are suitable for active thermography and the ability to locate subsurface defects on composites. An excitation source in multiple 250 W lamps is mounted onto a UAV and is solely battery powered with a remote trigger to power cycle them. Multiple experiments address the interference from the UAV whilst performing an active IRT inspection. The optimal distances and time required for a UAV inspection using IRT are calculated. Multiple signal processing techniques are used to analyse the composites which help locate the sub-surface defects. It was observed that a UAV can successfully carry the required sensors and equipment for an Active thermographic NDT inspection which can provide access to difficult areas. Most active thermographic inspection equipment is large, heavy, and expensive. Furthermore, using such equipment for the inspection of complex structures is time-consuming. For example, a cherry picker would be required to inspect the tail of an aircraft. This solution looks to assist engineers in inspecting complex composite structures and could potentially significantly reduce the time and cost of a routine inspection. Engineering and Physical Sciences Research Council (EPSRC): EP/N509450/1 and Innovate UK: 105625.

An effective implementation of enhanced sampling algorithms for molecular dynamics simulations requires a priori knowledge of the approximate reaction coordinate describing the relevant mechanisms in the system. Here we demonstrate how the artificial intelligence based recent State Predictive Information Bottleneck (SPIB) approach can learn such a reaction coordinate as a deep neural network even from under-sampled trajectories. We demonstrate its usefulness by achieving more than 40 magnitudes of acceleration in simulating two test-piece biophysical systems through well-tempered metadynamics performed by biasing along the SPIB learned reaction coordinate. These include left- to right- handed chirality transitions in a synthetic protein (Aib)(9), and permeation of a small, asymmetric molecule benzoic acid through a synthetic, symmetric phospholipid bilayer. In addition to significantly accelerating the dynamics and achieving back-and-forth movement between different metastable states, the SPIB based reaction coordinate gives mechanistic insight into the processes driving these two important problems.

Glutamate is the predominant excitatory neurotransmitter in the mammalian central nervous system (CNS). Excitatory Amino Acid Transporters (EAATs) regulate extracellular glutamate by transporting it into cells, mostly glia, to terminate neurotransmission and to avoid neurotoxicity. EAATs are also chloride (Cl−) channels, but the physiological role of Cl− conductance through EAATs is poorly understood. Mutations of human EAAT1 (hEAAT1) have been identified in patients with episodic ataxia type 6 (EA6). One mutation showed increased Cl− channel activity and decreased glutamate transport, but the relative contributions of each function of hEAAT1 to mechanisms underlying the pathology of EA6 remain unclear. Here we investigated the effects of five additional EA6-related mutations on hEAAT1 function in Xenopus laevis oocytes, and on CNS function in a Drosophila melanogaster model of locomotor behavior. Our results indicate that mutations with decreased hEAAT1 Cl− channel activity and functional glutamate transport can also contribute to the pathology of EA6, highlighting the importance of Cl− homeostasis in glial cells for proper CNS function. We also identified a novel mechanism involving an ectopic sodium (Na+) leak conductance in glial cells. Together, these results strongly support the idea that EA6 is primarily an ion channelopathy of CNS glia.

Structural Health Monitoring (SHM) aims to shift aircraft maintenance from a time-based to a condition-based approach. Within all the SHM techniques, Acoustic Emission (AE) allows for the monitoring of large areas by analyzing Lamb waves propagating in plate like structures. In this study, the authors proposed a Time Reversal (TR) methodology with the aim of reconstructing an original and unaltered signal from an AE event. Although the TR method has been applied in Narrow-Band (NwB) signal reconstruction, it fails when a Broad-Band (BdB) signal, such as a real AE event, is present. Therefore, a novel methodology based on the use of a Frequencies Compensation Transfer Function (FCTF), which is capable of reconstructing both NwB and real BdB signals, is presented. The study was carried out experimentally using several sensor layouts and materials with two different AE sources: (i) a Numerically Built Broadband (NBB) signal, (ii) a Pencil Lead Break (PLB). The results were validated numerically using Abaqus/CAETM with the implementation of absorbing boundaries to minimize edge reflections.

Excitatory amino acid transporters (EAATs) are glutamate transporters that belong to the solute carrier 1A (SLC1A) family. They couple glutamate transport to the co-transport of three sodium (Na+) ions and one proton (H+) and the counter-transport of one potassium (K+) ion. In addition to this coupled transport, binding of substrate and Na+ ions to EAATs activates a thermodynamically uncoupled chloride (Cl−) conductance. Structures of SLC1A family members have revealed that these transporters use a twisting elevator mechanism of transport, where a mobile transport domain carries substrate and coupled ions across the membrane, while a static scaffold domain anchors the transporter in the membrane. We have recently demonstrated that the uncoupled Cl− conductance is activated by the formation of an aqueous pore at the domain interface during the transport cycle in archaeal GltPh. However, a pathway for the uncoupled Cl− conductance has not been reported for the EAATs and it is unclear if such a pathway is conserved. Here, we employ all-atom molecular dynamics (MD) simulations combined with enhanced sampling, free-energy calculations, and experimental mutagenesis to approximate large-scale conformational changes during the transport process and identified a Cl− conducting conformation in human EAAT1. We were able to extensively sample the large-scale structural transitions, allowing us to capture an intermediate conformation formed during the transport cycle with a continuous aqueous pore at the domain interface. The free-energy calculations performed for the conduction of Cl− and Na+ ions through the captured conformation, highlight the presence of two hydrophobic gates which control the selective movement of Cl− through the aqueous pathway. Overall, our findings provide insights into the mechanism by which a human glutamate transporter can support the dual functions of active transport and passive Cl− permeation and confirming the commonality of this mechanism in different members of the SLC1A family.

Phosphatidylinositol-4,5-bisphosphate (PIP2) is a signaling lipid which regulates voltage-gated Kv7/KCNQ potassium channels. Altered PIP2 sensitivity of neuronal Kv7.2 channel is involved in KCNQ2 epileptic encephalopathy. However, the molecular action of PIP2 on Kv7.2 gating remains largely elusive. Here, we use molecular dynamics simulations and electrophysiology to characterize PIP2 binding sites in a human Kv7.2 channel. In the closed state, PIP2 localizes to the periphery of the voltage-sensing domain (VSD). In the open state, PIP2 binds to 4 distinct interfaces formed by the cytoplasmic ends of the VSD, the gate, intracellular helices A and B and their linkers. PIP2 binding induces bilayer-interacting conformation of helices A and B and the correlated motion of the VSD and the pore domain, whereas charge-neutralizing mutations block this coupling and reduce PIP2 sensitivity of Kv7.2 channels by disrupting PIP2 binding. These findings reveal the allosteric role of PIP2 in Kv7.2 channel activation.

Using aerial platforms for Non-Destructive Inspection (NDI) of large and complex structures is a growing field of interest in various industries. Infrastructures such as: buildings, bridges, oil and gas, etc. refineries require regular and extensive inspections. The inspection reports are used to plan and perform required maintenance, ensuring their structural health and the safety of the workers. However, performing these inspections can be challenging due to the size of the facility, the lack of easy access, the health risks for the inspectors, or several other reasons, which has convinced companies to invest more in drones as an alternative solution to overcome these challenges. The autonomous nature of drones can assist companies in reducing inspection time and cost. Moreover, the employment of drones can lower the number of required personnel for inspection and can increase personnel safety. Finally, drones can provide a safe and reliable solution for inspecting hard-to-reach or hazardous areas. Despite the recent developments in drone-based NDI to reliably detect defects, several limitations and challenges still need to be addressed. In this paper, a brief review of the history of unmanned aerial vehicles, along with a comprehensive review of studies focused on UAV-based NDI of industrial and commercial facilities, are provided. Moreover, the benefits of using drones in inspections as an alternative to conventional methods are discussed, along with the challenges and open problems of employing drones in industrial inspections, are explored. Finally, some of our case studies conducted in different industrial fields in the field of Non-Destructive Inspection are presented.

Fatty acid (FA) transfer proteins extract FA from membranes and sequester their ligand to facilitate its movement through the cytosol. While detailed views of soluble protein-FA complexes are available, how FA exchange occurs at the membrane has remained unknown. Staphylococcus aureus FakB1 is a prototypical bacterial FA transfer protein that binds palmitate within a narrow, buried tunnel. Here, we determine the conformational change from this closed state to an open state that engages the phospholipid bilayer. Upon membrane binding, a dynamic loop in FakB1 that covers the FA binding site disengages and folds into an amphipathic helix. This helix inserts below the phosphate plane of the bilayer to create a diffusion channel for the FA to exchange between the protein and the membrane. The structure of the bilayer-associated conformation of FakB1 has local similarities with mammalian FA binding proteins and provides a general conceptual framework for how these proteins interact with the membrane to promote lipid transfer.

Phosphatidylinositol-4,5-bisphosphate (PIP2) is a signaling lipid which regulates voltage-gated Kv7/KCNQ potassium channels. Altered PIP2 sensitivity of neuronal Kv7.2 channel is involved in KCNQ2 epileptic encephalopathy. However, the molecular action of PIP2 on Kv7.2 gating remains largely elusive. Here, we use molecular dynamics simulations and electrophysiology to characterize PIP2 binding sites in a human Kv7.2 channel. In the closed state, PIP2 localizes to the periphery of the voltage-sensing domain (VSD). In the open state, PIP2 binds to 4 distinct interfaces formed by the cytoplasmic ends of the VSD, the gate, intracellular helices A and B and their linkers. PIP2 binding induces bilayer-interacting conformation of helices A and B and the correlated motion of the VSD and the pore domain, whereas charge-neutralizing mutations block this coupling and reduce PIP2 sensitivity of Kv7.2 channels by disrupting PIP2 binding. These findings reveal the allosteric role of PIP2 in Kv7.2 channel activation.

Diagnosis and prognosis of failures for aircrafts’ integrity are some of the most important regular functionalities in complex and safety-critical aircraft structures. Further, development of failure diagnostic tools such as Non-Destructive Testing (NDT) techniques, in particular, for aircraft composite materials, has been seen as a subject of intensive research over the last decades. The need for diagnostic and prognostic tools for composite materials in aircraft applications rises and draws increasing attention. Yet, there is still an ongoing need for developing new failure diagnostic tools to respond to the rapid industrial development and complex machine design. Such tools will ease the early detection and isolation of developing defects and the prediction of damages propagation; thus allowing for early implementation of preventive maintenance and serve as a countermeasure to the potential of catastrophic failure. This paper provides a brief literature review of recent research on failure diagnosis of composite materials with an emphasis on the use of active thermography techniques in the aerospace industry. Furthermore, as the use of unmanned aerial vehicles (UAVs) for the remote inspection of large and/or difficult access areas has significantly grown in the last few years thanks to their flexibility of flight and to the possibility to carry one or several measuring sensors, the aim to use a UAV active thermography system for the inspection of large composite aeronautical structures in a continuous dynamic mode is proposed., Thermosense: Thermal Infrared Applications XLIII, April 12-16, 2021, Online Only, United States, Series: Proceedings of SPIE

Piezoelectric lead zirconate titanate (PZT) sensors are widely used in various structural health monitoring (SHM) applications, where data acquired by the PZT sensors are used for damage detection. Any failure of the PZT sensors will have a detrimental effect in the ability of SHM systems to detect damage. Therefore, detecting faulty PZT sensor is critical to reduce any false-calls associated with malfunctioning sensor to ensure proper functionality of SHM systems. This paper proposes a self-diagnostic method to monitoring the health of PZT sensors using the electro-mechanical impedance (EMI) data in two steps. In the first detection step, the onedimensional convolutional autoencoder (1D-CAE) is employed to obtain the reconstruction error as anomaly scores from the raw EMI data. Hence, the faulty PZT sensors can be detected by comparing the anomaly score with a pre-defined threshold. In the second diagnostic step, the data feature is first extracted with the 1D-CAE. The extracted feature is then fed into a multilayer perceptron (MLP) classifier to classify the fault type of the PZT sensor. The proposed method was validated through experiments, where typical in-service induced damages such as impact, environmental effect, sensor breakage localized high temperature heating, etc. were introduced. The results demonstrate the effectiveness of the proposed method for both detection and diagnosis of various types of PZT sensor damage., Health Monitoring of Structural and Biological Systems XV, March 22-27, 2021, United States, Series: Proceedings of SPIE

Phospholipid extrusion by ABC subfamily A (ABCA) exporters is central to cellular physiology, although the specifics of the underlying substrate interactions and transport mechanisms remain poorly resolved at the molecular level. Here we report cryo-EM structures of lipid-embedded human ABCA7 in an open state and a nucleotide-bound, closed state at resolutions between 3.6-4.0 Å. The former reveals an ordered patch of bilayer lipids traversing the transmembrane domain (TMD), while the latter reveals a lipid-free, closed TMD with a small extracellular opening. These structures offer a structural framework for both substrate entry and exit from the ABCA7 TMD and highlight conserved rigid-body motions that underlie the associated conformational transitions. Combined with functional analysis and molecular dynamics (MD) simulations, our data also shed light on lipid partitioning into the ABCA7 TMD and localized membrane perturbations that underlie ABCA7 function and have broader implications for other ABCA family transporters.

Unmanned Aerial Vehicles (UAVs) that can fly around an aircraft carrying several sensors, e.g., thermal and optical cameras, to inspect the parts of interest without removing them can have significant impact in reducing inspection time and cost. One of the main challenges in the UAV based active InfraRed Thermography (IRT) inspection is the UAV’s unexpected motions. Since active thermography is mainly concerned with the analysis of thermal sequences, unexpected motions can disturb the thermal profiling and cause data misinterpretation especially for providing an automated process pipeline of such inspections. Additionally, in the scenarios where post-analysis is intended to be applied by an inspector, the UAV’s unexpected motions can increase the risk of human error, data misinterpretation, and incorrect characterization of possible defects. Therefore, post-processing is required to minimize/eliminate such undesired motions using digital video stabilization techniques. There are number of video stabilization algorithms that are readily available, however, selecting the best suited one is also challenging. Therefore, this paper evaluates video stabilization algorithms to minimize/mitigate undesired UAV motion and proposes a simple method to find the best suited stabilization algorithm as a fundamental first step towards a fully operational UAV-IRT inspection system.

Approximately up to one-fifth of the direct operating cost of a commercial civilian fixed-wing aircraft is projected to be due to inspection and maintenance alone. Managing aircraft health with minimal human intervention and technologies that can perform continuous or on-demand monitoring/evaluation of aircraft components without having to take the aircraft out of service can have a significant impact on increasing availability while reducing maintenance cost. The ambition of these monitoring technologies is to shift aircraft maintenance practice from planned maintenance (PM), where the aircraft is taken out of service for scheduled inspection/maintenance, to condition-based maintenance (CBM), where aircraft is taken out of service only when maintenance is required, while maintaining the required levels of safety. Structural health monitoring (SHM) techniques can play a vital role in progressing towards CBM practice. Therefore, this chapter aims to provide the reader with a brief overview of the different SHM techniques and their use, as well as, challenges in implementing them for aircraft applications., Series: Springer Aerospace Technology

This chapter provides an overview of the common types of defects found in various structural materials and joints in aircraft. Materials manufacturing methods (including large-scale production) have been established in the aircraft industry. However, as will be seen in this chapter, manufacturing defects and defects during in-service conditions are very common across all material types. The structural material types include metals, composites, coatings, adhesively bonded and stir-welded joints. This chapter describes the defect types as a baseline for the description of their detection with the methods of Chap. 5 to 8. Based on the understanding of the defect types, there is great expectation for a technical breakthrough for the application of structural health monitoring (SHM) damage detection systems, where continuous monitoring and assessment with high throughput and yield will produce the desired structural integrity., Series: Springer Aerospace Technology

Phosphatidylinositol-4,5-bisphosphate (PIP2) is a signaling lipid which regulates voltage-gated Kv7/KCNQ potassium channels. Altered PIP2 sensitivity of neuronal Kv7.2 channel is involved in KCNQ2 epileptic encephalopathy. However, the molecular action of PIP2 on Kv7.2 gating remains largely elusive. Here, we use molecular dynamics simulations and electrophysiology to characterize PIP2 binding sites in a human Kv7.2 channel. In the closed state, PIP2 localizes to the periphery of the voltage-sensing domain (VSD). In the open state, PIP2 binds to 4 distinct interfaces formed by the cytoplasmic ends of the VSD, the gate, intracellular helices A and B and their linkers. PIP2 binding induces bilayer-interacting conformation of helices A and B and the correlated motion of the VSD and the pore domain, whereas charge-neutralizing mutations block this coupling and reduce PIP2 sensitivity of Kv7.2 channels by disrupting PIP2 binding. These findings reveal the allosteric role of PIP2 in Kv7.2 channel activation., Pant et al. describe the mechanism by which PIP2 might regulate homomeric Kv7.2 channels. They identify sites important in the binding of the PIP2 lipid to Kv7.2 channels and propose that the PIP2 binding to a specific site results in the coupling between the voltage sensor domain (VSD) and pore domain (PD), which stabilizes the open state of the channel.

Artificial intelligence (AI) based approaches have had indubitable impact across the sciences through the ability to extract relevant information from raw data. Recently AI has also seen use for enhancing the efficiency of molecular simulations, wherein AI derived slow modes are used to accelerate the simulation in targeted ways. However, while typical fields where AI is used are characterized by a plethora of data, molecular simulations per construction suffer from limited sampling and thus limited data. As such the use of AI in molecular simulations can suffer from a dangerous situation where the AI optimization could get stuck in spurious regimes, leading to incorrect characterization of the reaction coordinate (RC) for the problem at hand. When such an incorrect RC is then used to perform additional simulations, one could start to deviate progressively from the ground truth. To deal with this problem of spurious AI solutions, here we report a novel and automated algorithm using ideas from statistical mechanics. It is based on the notion that a more reliable AI solution for many problems in chemistry and biophysics will be one that maximizes the time scale separation between slow and fast processes. To learn this timescale separation even from limited data, we use a maximum path entropy or caliber-based framework. We show the applicability of this automatic protocol for 3 classic benchmark problems, namely capturing the conformational dynamics of a model peptide (alanine dipeptide), ligand unbinding dynamics from a protein, and extensive sampling of the folding/unfolding energy landscape of the C-terminal domain of protein G (GB1-C16). We believe our work will lead to increased and robust use of trustworthy AI in molecular simulations of complex systems.

Glutamate is the most abundant excitatory neurotransmitter in the central nervous system, therefore its precise control is vital for maintaining normal brain function and preventing excitotoxicity1. Removal of extracellular glutamate is achieved by plasma membrane-bound transporters, which couple glutamate transport to sodium, potassium and pH gradients using an elevator mechanism2–5. Glutamate transporters also conduct chloride ions via a channel-like process that is thermodynamically uncoupled from transport6–8. However, the molecular mechanisms that allow these dual-function transporters to carry out two seemingly contradictory roles are unknown. Here we report the cryo-electron microscopy structure of a glutamate transporter homologue in an open-channel state, revealing an aqueous cavity that is formed during the transport cycle. Using functional studies and molecular dynamics simulations, we show that this cavity is an aqueous-accessible chloride permeation pathway gated by two hydrophobic regions, and is conserved across mammalian and archaeal glutamate transporters. Our findings provide insight into the mechanism by which glutamate transporters support their dual function and add a crucial piece of information to aid mapping of the complete transport cycle shared by the SLC1A transporter family.

Proton-coupled transporters use transmembrane proton gradients to power active transport of nutrients inside the cell. High-resolution structures often fail to capture the coupling between proton and ligand binding, and conformational changes associated with transport. We combine HDX-MS with mutagenesis and MD simulations to dissect the molecular mechanism of the prototypical transporter XylE. We show that protonation of a conserved aspartate triggers conformational transition from outward-facing to inward-facing state. This transition only occurs in the presence of substrate xylose, while the inhibitor glucose locks the transporter in the outward-facing state. MD simulations corroborate the experiments by showing that only the combination of protonation and xylose binding, and not glucose, sets up the transporter for conformational switch. Overall, we demonstrate the unique ability of HDX-MS to distinguish between the conformational dynamics of inhibitor and substrate binding, and show that a specific allosteric coupling between substrate binding and protonation is a key step to initiate transport., SCOPUS: ar.j, info:eu-repo/semantics/published

Kv7 channels are enriched at the axonal plasma membrane where their voltage-dependent potassium currents suppress neuronal excitability. Mutations in Kv7.2 and Kv7.3 subunits cause epileptic encephalopathy (EE), yet the underlying pathogenetic mechanism is unclear. Here, we used novel statistical algorithms and structural modeling to identify EE mutation hotspots in key functional domains of Kv7.2 including voltage sensing S4, the pore loop and S6 in the pore domain, and intracellular calmodulin-binding helix B and helix B-C linker. Characterization of selected EE mutations from these hotspots revealed that L203P at S4 induces a large depolarizing shift in voltage dependence of Kv7.2 channels and L268F at the pore decreases their current densities. While L268F severely reduces expression of heteromeric channels in hippocampal neurons without affecting internalization, K552T and R553L mutations at distal helix B decrease calmodulin-binding and axonal enrichment. Importantly, L268F, K552T, and R553L mutations disrupt current potentiation by increasing phosphatidylinositol 4,5-bisphosphate (PIP2), and our molecular dynamics simulation suggests PIP2 interaction with these residues. Together, these findings demonstrate that each EE variant causes a unique combination of defects in Kv7 channel function and neuronal expression, and suggest a critical need for both prediction algorithms and experimental interrogations to understand pathophysiology of Kv7-associated EE.

Fatty acid (FA) transfer proteins extract FA from membranes and sequester them to facilitate their movement through the cytosol. Detailed structural information is available for these soluble protein-FA complexes, but the structure of the protein conformation responsible for FA exchange at the membrane is unknown. Staphylococcus aureus FakB1 is a prototypical bacterial FA transfer protein that binds palmitate within a narrow, buried tunnel. Here, we define the conformational change from a "closed" FakB1 state to an "open" state that associates with the membrane and provides a path for entry and egress of the FA. Using NMR spectroscopy, we identified a conformationally flexible dynamic region in FakB1, and X-ray crystallography of FakB1 mutants captured the conformation of the open state. In addition, molecular dynamics simulations show that the new amphipathic α-helix formed in the open state inserts below the phosphate plane of the bilayer to create a diffusion channel for the hydrophobic FA tail to access the hydrocarbon core and place the carboxyl group at the phosphate layer. The membrane binding and catalytic properties of site-directed mutants were consistent with the proposed membrane docked structure predicted by our molecular dynamics simulations. Finally, the structure of the bilayer-associated conformation of FakB1 has local similarities with mammalian FA binding proteins and provides a conceptual framework for how these proteins interact with the membrane to create a diffusion channel from the FA location in the bilayer to the protein interior.

Nondestructive testing and evaluation (NDT&E) are commonly used in the industry for their ability to identify damage and assess material conditions. Ultrasonic testing (UT) is one of the most popular NDT&E techniques. A variant of ultrasonic testing known as nonlinear ultrasonic testing (NUT) has some advantages over conventional (linear) UT as it is more sensitive to damages in their early stages; even at the microscopic levels. Furthermore, the nonlinear characteristics of ultrasonic waves can be correlated to several material properties. In the last two decades, the NUT method has been investigated from two aspects, namely the direct (modeling) problem and the inverse (NUT testing) problem. The direct problem aims to establish the nonlinear mechanism and analyze the behavior of wave-damage interaction. The inverse problem is investigated under three headings: (1) data acquisition with NUT techniques, (2) signal pre-processing and feature extraction, and (3) parameter analysis for damage characterization. The conventional data analytical methods extract nonlinear features from noisy signals and build a damage index to characterize damages. However, damage index-based analyzing model can be challenging, as other factors affect the overall system nonlinearity such as complex specimen geometry, different damage characteristics, varying ambient conditions, and measurement uncertainties. To overcome these shortcomings, machine learning (ML) methods appear promising for the analysis of complex nonlinear ultrasonic signals by exploiting data mining and pattern recognition capabilities. Therefore, this paper aims to provide a comprehensive review of the state-of-the-art ML-enriched NUT for damage characterization. Other NUT-based technologies are also reviewed, including modeling of wave-damage interaction, different NUT techniques for data acquisition, signal pre-processing methods, and damage index-based parameter analysis strategies for damage characterization. Major emphasis is placed on the application of ML methods for NDT&E applications. Additionally, future research trends on data augmentation, complex damage characterization, and baseline-free methods using NUT are also discussed.

Membrane transporters are key gatekeeper proteins at cellular membranes that closely control the traffic of materials. Their function relies on structural rearrangements of varying degrees that facilitate substrate translocation across the membrane. Characterizing these functionally important molecular events at a microscopic level is key to our understanding of membrane transport, yet challenging to achieve experimentally. Recent advances in simulation technology and computing power have rendered molecular dynamics (MD) simulation a powerful biophysical tool to investigate a wide range of dynamical events spanning multiple spatial and temporal scales. Here, we review recent studies of diverse membrane transporters using computational methods, with an emphasis on highlighting the technical challenges, key lessons learned, and new opportunities to illuminate transporter structure and function.

The pleckstrin homology (PH) domain of general receptor for phosphoionositides 1 (GRP1-PHD) binds specifically to phosphatidylinositol (3,4,5)-triphosphate (PIP3 ), and acts as a second messenger. Using an extensive array of molecular dynamics (MD) simulations employing highly mobile membrane mimetic (HMMM) model as well as complementary full membrane simulations, we capture differentiable binding and dynamics of GRP1-PHD in the presence of membranes containing PC, PS, and PIP3 lipids in varying compositions. While GRP1-PHD forms only transient interactions with pure PC membranes, incorporation of anionic lipids resulted in stable membrane-bound configurations. We report the first observation of two distinct PIP3 binding modes on GRP1-PHD, involving PIP3 interactions at a "canonical" and at an "alternate" site, suggesting the possibility of simultaneous binding of multiple anionic lipids. The full membrane simulations confirmed the stability of the membrane bound pose of GRP1-PHD as captured from our HMMM membrane binding simulations. By performing additional steered membrane unbinding simulations and calculating nonequilibrium work associated with the process, as well as metadynamics simulations, on the protein bound to full membranes, allowing for more quantitative examination of the binding strength of the GRP1-PHD to the membrane, we demonstrate that along with the bound PIP3 , surrounding anionic PS lipids increase the energetic cost of unbinding of GRP1-PHD from the canonical mode, causing them to dissociate more slowly than the alternate mode. Our results demonstrate that concurrent binding of multiple anionic lipids by GRP1-PHD contributes to its membrane affinity, which in turn control its signaling activity. © 2019 Wiley Periodicals, Inc.

Acoustic Emission (AE) monitoring can be used to detect and locate structural damage such as growing fatigue cracks. The accuracy of damage location and consequently the inference of its significance for damage assessment is dependent on the wave propagation properties in terms of wave velocity, dispersion, attenuation and wave mode conversion. These behaviors are understood and accounted for in simplistic structures, however, actual structures are geometrically complex, with components comprising of different materials. One of the key challenges in such scenarios is the ability to positively identify wave modes and correctly associate their properties for damage location analysis. In this study, a novel method for wave mode identification is presented based on phase and instantaneous frequency analysis. Finite Element (FE) simulations and experiments on a representative aircraft wing structure were conducted to evaluate the performance of the technique. The results show how a phase analysis obtained from a Hilbert Transform of the wave signal in combination with variations of the instantaneous frequency of the wave signal, can be used to determine the arrival and therefore identification of the different wave modes on a complex structure. The methodology outlined in this paper was proven on an Automatic Sensor Test wave signal, Pencil Lead Breaks and Hanning windows and it was shown that the percentage difference is between 3% and 15% for the A0 and S0 wave speed respectively.

The cellular membrane constitutes one of the most fundamental compartments of a living cell, where key processes such as selective transport of material and exchange of information between the cell and its environment are mediated by proteins that are closely associated with the membrane. The heterogeneity of lipid composition of biological membranes and the effect of lipid molecules on the structure, dynamics, and function of membrane proteins are now widely recognized. Characterization of these functionally important lipid-protein interactions with experimental techniques is however still prohibitively challenging. Molecular dynamics (MD) simulations offer a powerful complementary approach with sufficient temporal and spatial resolutions to gain atomic-level structural information and energetics on lipid-protein interactions. In this review, we aim to provide a broad survey of MD simulations focusing on exploring lipid-protein interactions and characterizing lipid-modulated protein structure and dynamics that have been successful in providing novel insight into the mechanism of membrane protein function.

We investigate the role of the length scale of attraction on the dynamical heterogeneity in three-dimensional core-softened (CS) model liquid having interaction potential of two different length scales. Using the simple CS model, we have shown that the presence of heterogeneity is attributed to the existence of attractive interactions in a system. The time scale associated with the peak position of the non-Gaussian parameter is found to be strongly dependent on the temperature for different length scale of attraction. The dynamical heterogeneity dramatically decreases with the increase in temperature. We further characterize dynamical heterogeneity by calculating new non-Gaussian parameter which indicates the existence of much longer relaxation timescale than that suggested by the peak position in the non-Gaussian parameter. Furthermore, we have shown the presence of particles, whose displacement distribution deviates from the Gaussian distribution and contributes towards the origin/presence of heterogeneity. Dynamical heterogeneity strongly depends on the length scale as well as the strength of attraction. System A which does not show that dynamical heterogeneity corresponds to repulsive interaction with large interaction length scale. System B which corresponds to attractive interaction with a small length scale of interaction shows dynamical heterogeneity. Non-Gaussian parameter $$[\alpha _2 ( t )]$$ for system A and system B at different temperatures