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Poly ADP-ribose polymerase-1 (PARP-1), due to its role in DNA damage and repair, has been identified as a crucial therapeutic target to attenuate cancer development and progression. More so, selective inhibition has remained a focal point in PARP-1 targeting and has led to the development of numerous compounds, including the recently identified Cpd10n, a novel homoerythrina alkaloid derivative. To expound on the selective PARP-1 inhibition mechanisms by Cpd10n, we employed computational simulation methods in this study. Findings revealed that the inhibitor stabilized the characteristic motion of activated PARP-1 as evidenced by reductions in residual deviations and structural flexibility. Findings further revealed that Cpd10n was favorably bound at the active site PARP-1 as supported by the occurrence of strong hydrogen and halogen bonds based on complementarity. These were in addition to aromatic bonds with an enhanced ring to ring stability. Steady and high-affinity interactions between the fluorine atom of Cpd10n and Glu988 could potentiate the selective activity of the compound. Interaction analyses also revealed that inhibitor binding was strongly dependent on electrostatic effects over van der Waal contributions, which were relatively minimal. We believe findings from this study will further contribute to the rational structure-based design of highly selective PARP-1 inhibitors.

Aims: Design isoxazole bearing leads as dual inhibitors against Amyloid β and BACE-1 loop in protein fibrillation. Background: Protein fibrillation is one of the key reasons for several diseases, namely Alzheimer’s, Parkinson’s, and many others. One of the key strategies of preventing protein fibrillation is destabilizing the protein fibrils themselves or inhibiting the amyloid fibril-forming pathway in the initial stage. Introduction: Attempts have been taken to design newer leads to inhibit protein fibrillation by targeting the β-amyloidogenesis pathway in the brain. To exploit interfenestration between Amyloid β -42 protein and BACE-1 (β-site amyloid precursor protein cleaving enzyme) for amyloidogenesis, studies are undertaken to design dual inhibitors against the same. Method: In vitro binding interactions were found using docking, de novo ligand design, and MD simulation study. Results: Three compounds bearing an isoxazole heterocyclic nucleus were designed which could successfully bind to the hydrophobic raft and salt bridge residues Asp 23-Lys-26 of Amyloid β, destabilizing the growing fibril. Additionally, one of our candidate compounds exhibited force of interaction with Thr232 at the S3 pocket of BACE-1, interacted with key residue Asp228, Tyr71, and Thr72 of the β-hairpin flap and hydrogen bonding with Gly11 at loop 10s. Conclusion: Protein flexibility dynamics of the Aβ-42 protein revealed that there is a considerable conformational change of the same with or without ligand binding. The lower RMSF of the bound region and reprogramming residual contacts within the Aβ-42 protein suggested successful binding of the ligand with the protein, lowering the access for further β-β dimerization.

Non-small cell lung cancer with an activating epidermal growth factor receptor (EGFR) mutation responds well to targeted drugs. In most cases, drug resistance appears after about a year. Several studies have been conducted on the kinase domain of EGFR to understand the drug resistance mechanism. Since EGFR is a multi-domain protein, mutation in the kinase domain may affect the other domains as well. In this study, we examine the complete structure of the multi-domain EGFR protein and its mutants. We performed molecular dynamics simulations for wildtype EGFR, EGFR with L858R mutation, and EGFR with L858R and T790M mutations. We applied normal mode analysis and complex network analysis to extract the correlated motions in the domains of EGFR. The normal modes are used to construct the dynamic cross-correlation map (DCCM). Simulation results show different patterns of correlated motions in each domain of EGFR mutants compared to the wildtype. In Domains 1 and 3 of the extracellular region, a small number of weak positively correlated motions are extracted. Domains 2 and 4 show large numbers of both positive and negative motions. However, the negatively correlated motions are stronger in mutant structures compared to the wildtype. In Domain 7, some residues showed a positive correlation around the main diagonal. We also identified different communities, nodes and crucial residues in the domains of the structures, which can be important for the function of EGFR. Moreover, hydrogen bond analysis is performed for the stability analysis. The mutant structures have fewer hydrogen bonds compared to the wildtype. Overall, these findings are useful for understanding the dynamics and communications in EGFR domains.

Copper hexadecafluorophthalocyanine (CuFPc) has excellent environmental and thermal stability and stable n-type semiconducting properties under ambient conditions. We investigated the exciton diffu...

Because of the lack of facile and accurate methods to track stress granule (SG) dynamics in live cells and in vivo, in-depth studies of the biological roles of this attractive membraneless organelle have been limited. Herein, we report the first small-molecule probe, TASG, for the selective, convenient and real-time monitoring of SGs. This novel molecule can simultaneously bind to SG RNAs, the core SG protein G3BP1, and their complexes, triggering a significant enhancement in fluorescence intensity, making TASG broadly applicable to SG imaging under various stress conditions in fixed and live cells, ex vivo and in vivo. Using TASG, the complicated endogenous SG dynamics were revealed in both live cells and C. elegans. Collectively, our work provides an ideal probe that has thus far been absent in the field of SG investigations. We anticipate that this powerful tool may create exciting opportunities to investigate the underlying roles of SGs in different organisms.

Butyl acrylate polymer networks were synthesized to understand the effect of permanent cross-links on large, anisotropic dye diffusion. The average degree of polymerization between cross-links (Nx)...

The force- and flow-induced translocation processes of linear and ring polymers are studied using a combination of multiparticle collision dynamics and molecular dynamics, focusing on the behavior of the polymer translocation time. We compare the force- and flow-induced translocations of linear and ring polymers. It is found that when the translocation time (τ*) is characterized by scaling exponents, δ, δ', and α, via the relations τ* ∼ fδNα and τ* ∼ Jδ'Nα, the scaling exponents are not constants. For long chains tested, α = 1.0 for both force- and flow-induced translocations. The difference between the force- and flow-induced translocations stems from different monomer crowding effects due to distinct flow patterns outside the channel. Furthermore, general relations for polymer translocation time are derived for these two translocation processes, which are in good agreement with the simulation results. Our results provide clear molecular pictures for the force- and flow-induced translocations, which shed light on the understanding of translocation dynamics and provide guidance for practical applications such as molecular sequencing and ultrafiltration.

The time-variant matrix inversion (TVMI) problem solving is the hotspot of current research because of its frequent appearance and application in scientific research and industrial production. The generalized inverse problem of singular square matrix and nonsquare matrix can be related to Penrose equations (PEs). The PEs implicitly define the generalized inverse of a known matrix, which is of fundamental theoretical significance. Therefore, the in-depth study of PEs might enlighten problem solving of TVMI in a foreseeable way. For the first time, we construct three different matrix error-monitoring functions based on PEs and propose the corresponding models for TVMI problem solving by using the substitution technique and ZNN design formula. In order to facilitate computer simulation, the obtained continuous-time models are discretized by using ZTD (Zhang time discretization) formulas. Furthermore, the feasibility and effectiveness of the novel Zhang neural network (ZNN) multiple-multiplication model for matrix inverse (ZMMMI) and the PEs-based Getz–Marsden dynamic system (PGMDS) model in solving the problem of TVMI are investigated and shown via theoretical derivation and computer simulation. Computer experiment results also illustrate that the direct derivative dynamics model for TVMI is less effective and feasible.

The Golgi apparatus and membrane tubules derived from this organelle play essential roles in membrane trafficking in eukaryotic cells. High-resolution live cell imaging is one highly suitable method for studying the molecular mechanisms of dynamics of organelles during membrane trafficking events. Due to the complex morphological changes and dynamic movements of the Golgi apparatus and associated membrane tubules during membrane trafficking, it is challenging to accurately quantify them. In this study, a semi-automated 2D tracking system, 2D-GolgiTrack, has been established for quantifying morphological changes and movements of Golgi elements, specifically encompassing the Golgi apparatus and its associated tubules, the fission and fusion of Golgi tubules, and the kinetics of formation of Golgi tubules and redistribution of the Golgi-associated protein Rab6A to the endoplasmic reticulum. The Golgi apparatus and associated tubules are segmented by a combination of Otsu's method and adaptive local normalization thresholding. Curvilinear skeletons and tips of skeletons of segmented tubules are used for calculating tubule length by the Geodesic method. The k-nearest neighbor is applied to search the possible candidate objects in the next frame and link the correct objects of adjacent frames by a tracking algorithm to calculate changes in morphological features of each Golgi object or tubule, e.g., number, length, shape, branch point and position, and fission or fusion events. Tracked objects are classified into morphological subtypes, and the Track-Map function of morphological evolution visualizes events of fission and fusion. Our 2D-GolgiTrack not only provides tracking results with 95% accuracy, but also maps morphological evolution for fast visual interpretation of the fission and fusion events. Our tracking system is able to characterize key morphological and dynamic features of the Golgi apparatus and associated tubules, enabling biologists to gain a greater understanding of the molecular mechanisms of membrane traffic involving this essential organelle. Graphical Abstract Overview of the semi-automated 2D tracking system. There are two main parts to the system, namely detection and tracking. The workflow process requires a raw sequence of images (a), which is filtered by the Gaussian filter method (c), and threshold intensity (b) to segment elements of Golgi cisternae (d) and tubules (e). Post-processing outputs are binary images of the cisternae area and tubule skeletons. The tubules are classified into three lengths, namely short, medium, and long tubules (f). Outputs of segmentation are calculated as morphological features (g). The tracking processing starts by loading the segmented outputs (h) and key-inputs of direction reference (i; (DR)) and interval setting of the start ((S)) and end ((E)) frame numbers (j). A tubule of interest is selected by the user (k; (GT

A filament bundle is a type of yarn, which is composed of nearly parallel and highly oriented polymer monofilaments. Due to its nonlinearity both in material constitutive properties and structure, the filament bundle possesses nonlinear viscoelastic properties. It is important to study the dynamic behavior of the filament bundle accurately during its high-speed movement. Therefore, an accurate expression of the constitutive relation of the filament bundle is an essential prerequisite for its dynamic simulation and analysis. Continued the previous study in Part I: modeling filament bundle method, in this paper, an approach was proposed to identify the equivalent dynamic constitutive parameters of the filament bundle considering frequency-dependent characteristics. Firstly, the identification formulas of the dynamic elastic modulus and viscoelastic coefficients were derived based on the Kelvin model. Then, a testing method of the cross-sectional parameters of the filament bundle under a certain tension was proposed, and the testing device was developed to obtain the area of the filament bundle; The dynamic loading test of the bundle filament was conducted in a DMA Q800 dynamic mechanical tester. Thirdly, the equivalent dynamic elastic modulus and viscoelastic coefficients were obtained through the experimental test. Finally, an analytical method was proposed to verify the correctness of experimental results through simulation. The results show that the excitation frequency has a significant influence on the dynamic elastic modulus and viscoelastic coefficient, and the curves of the equivalent dynamic elastic modulus and viscoelastic coefficient present nonlinear variation characteristics.

Cox protein plays a critical role in deciding the lytic-lysogenic switch of P2 enteric phages. This phenomenon makes Cox protein one of the most important candidates in developing novel phage-based...

To investigate the dynamics of the orexin 2 receptor, which is a class A G protein-coupled receptor, we recently performed several microsecond-scale molecular dynamics simulations of the wild-type protein, of a mutant that stabilizes the inactive state, and of constitutively active mutants of the class A G protein-coupled receptors. Herein, we review the results of these molecular dynamics simulations of the orexin 2 receptor. In these simulations, characteristic conformational changes were observed in the V309(6.40)Y mutant. The conformational changes were related to the outward movement of the transmembrane helix 6 and the inward movement of the transmembrane helix 7, which are common structural changes in the activation of G protein-coupled receptors. The index for the quantitative evaluation of the active and inactive states of class A G protein-coupled receptors and the mechanism of the inward movement of the transmembrane helix 7 were examined. In this review, we also discuss the activation mechanism by comparing the structures obtained from the molecular dynamics simulations with the structure of the active state of the orexin 2 receptor clarified by cryo-electron microscopy in the recent years.

We performed a detailed analysis of changes in the profiles of osmotic deformability using the method of gradient ektacytometry. Changes in all determinants that form the deformation properties of red blood cells in Wistar rats in the juvenile period and before puberty were determined. The dynamics of the formation of the rheological properties of the blood after birth is characterized by a wave-like change in the studied determinants. The changes are explained by adaptive reactions to extrauterine life as a result of hematopoiesis activation and the transition of the red bone marrow to a new level of functioning with the predominant replacement of physiological reticulocytosis in newborns with mature erythrocytes. The most critical period is from 10 days to 1 month after birth. Starting from the second month, the deformation parameters of erythrocytes are stabilized.

Alstonia scholaris is a biologically active and important Devil tree. The medicinal importance pharmacology importance in particulars antiulcer activity of Alstonic scholaris in albino rats were Analyzed and reported. The juice of Alstona scholaris to rats show a remarkable record with respect to ranitidine tablet (to cure ulcer). This result initiate the author to study chemo mimetic nature of plant material for the removal of heavy metal in synthetic solution also extended studies with modified Alstonia scholaris and Wrichitia Tinctoria. An adsorption of 96% in all the cases was observed. A linear correlation with R2 value of 0.977 was calculated from the linear plot of optical densities of Alstonia Scholaris and modified Alstonia Scholaris similar trend in adsorption was also noted. In the case of Wrightia Tinctoria instead of modified Alstonia Scholaris

The resistance during the frictional interaction of polymeric materials with metallic materials is characterized by a significant dependence on the dynamics of the motion inputs. In a metal-polymer friction pair, the static friction resistance during standstill under load depends on the rate of growth of the force causing the relative motion. Tribological tests of selected (polymer-metal) sliding pairs were carried out. The selected polymers were polyurethane (TPU), polysulfone (PSU), and silicone rubber (SI). They interacted with a pin made of normalized C45 steel under unitary pressure p = 0.5 MPa in dry friction conditions at different gradients of the force driving the relative motion (dF/dt = 0.1-20 [N/s]). The static friction coefficient of the selected sliding pairs was determined on the basis of the recorded static friction force values. The test results show a significant influence of the rate of increase in the motion driving force on the values of static friction resistance. This is mainly due to the viscoelastic properties of polymers.

The purpose of the study was to determine metabolic parameters (alpha-amylase, alanine aminotransferase and aspartate aminotransferase, gamaglutamyl transpeptidase, glycoproteins and chondroitin sulfates) at the stages of sodium glutamate intake and after withdrawal. Materials and methods. Studies were conducted on 65 white male rats of reproductive age (2.5-3 months); the experimental group received 70 mg/kg of sodium glutamate per live weight for 8 weeks. The control group of animals received a standard diet. Before drug withdrawal, rats were studied every week, after drug withdrawal the studies were performed every two weeks (10, 12, 14, 16 weeks). Results and discussion. The study of the enzyme activity of alpha-amylase showed that this index increased on the average 1.3-fold compared with the control values, and at the end of the period when the rats were fed with sodium glutamate (8 weeks), the index increased by 3.85 times, alanine aminotransferase – by 12.3 times, aspartate aminotransferase – by 1.4 times, gamaglutamyl transpeptidase – by 2.7 times, glycoproteins – by 1.4 times, chondroitin sulfate – by 1.2 times. After transferring the animals to a normal diet, no recovery of the indices was found. According to the studied indicators sodium glutamate has a toxic effect on the liver, pancreas with elements of systemic inflammation. At the stages of discontinuation of monosodium glutamate recovery of most of the studied biochemical markers is not established. Alanine aminotransferase, determined on the 16th day after the abolition of monosodium glutamate, was reduced by 1.33 times compared to the last 8 weeks of admission, gamaglutamyl transpeptidase after 10 weeks was reduced by 1.5 times, after 12 weeks – by 0.4 times, after 14 and 16 weeks, the values corresponded to the control values at 10 week, the alpha-amylase level was reduced by 1.26 times, at 16 weeks – by 2.29 times, but they did not reach the control values. At the end of the study (16 weeks), the level of chondroitin sulfates was increased compared to 8 weeks of feeding animals with glutamate sodium by 1.27 times, and relative to control – by 1.56 times. There was a decrease in glycoprotein content at 16 weeks compared with 8 weeks of observation by 1.3 times, but did not reach the level of intact animals. Conclusion. Thus, the results of serum biochemical studies of rats treated with monosodium glutamate indicated the development of intoxication and its effect on biochemical markers, which were reflected in the dynamics of enzyme activity, inflammation and fibrosis. Most biochemical markers (except gamaglutamyl transpeptidase and aspartate aminotransferase) at the end of the experiment did not reach the level of intact animals, which is apparently due to the chronicity of the pathological process. In rats after discontinuation of monosodium glutamate from 10 to 16 weeks of the experiment, changes in biochemical parameters were observed, which indicated a toxic effect, which was accompanied by the development of subacute inflammatory process. After the transfer of animals to a normal diet, recovery is not established. Sodium glutamate according to the studied parameters has a toxic effect on the liver, pancreas with elements of systemic inflammation

The connection between slow activated relaxation in glass-forming liquids and various equilibrium thermodynamic properties remains intensely debated. The microscopic elastically collective nonlinea...

Elastic network model simulations were performed to investigate the conformational changes of MDM2 protein induced by its native substrate p53 and two small molecule inhibitor (NVP-CGM097 and HDM 201) bindings. Residues Phe 19, Trp 23, Leu 26 were observed to reside in the minima of slowest modes of p53, pointing to the accepted three finger binding model. Pro 27 displays the most significant hinge present in p53 and comes out to be another functionally important residue. Three distinct conserved regions are identified in MDM2. Regions I (residues 50–77) and III (residues 90–105) correspond to the binding interface of MDM2 including [Formula: see text] helix-2 ([Formula: see text]), Loop-2 (L2) and [Formula: see text] helix-4 ([Formula: see text]) domains which are stabilized during complex formation. Region II (residues 77–90) is a highly flexible region in both unbound and bound forms exhibiting high amplitude collective motion. MDM2 exhibits a scattered profile in the fastest modes of motion, while binding of p53 and inhibitors puts restraints on MDM2 pointing to induced-fit mechanism. Flexible docking using AutoDock Vina is performed to account for the flexible nature of the receptor and to elucidate the essential interactions in the binding cleft. [Formula: see text] domain controls the size of the cleft by keeping the cleft narrow in unbound MDM2; and open in the bound states. Inhibitors studied in this work (NVP-CGM097 and HDM201), which are recently undergoing clinical trials, succeed in mimicking p53 behavior which would shed light on the rational design of novel anticancer drugs.

Deposition based direct radon and thoron progeny sensors give a more accurate estimate of the inhalation dose due to radon and thoron. The dependence of equilibrium factor on aerosol concentration and the ventilation rates has been studied. Deposited flux represents a better index of inhalation dose. Deposition based sensors are based on deposition velocity of the progeny atoms in the environment which show variability only in extremities like very low aerosol environment or very high ventilation rates, which has been presented in this work. Wire-mesh capped deposition sensor which is based on attached fraction deposition velocity, has been studied at different particle concentrations and it can be used for such occupational environments.

Various nuclear magnetic resonance (NMR) methods are combined to study the structure and dynamics of Li1.5Al0.5Ti1.5(PO4)3 (LATP) samples, which were obtained from sintering at various temperatures between 650 and 900 °C. 6Li, 27Al, and 31P magic angle spinning (MAS) NMR spectra show that LATP crystallites are better defined for higher calcination temperatures. Analysis of 7Li spin-lattice relaxation and line-shape changes indicates the existence of two species of lithium ions with clearly distinguishable jump dynamics, which can be attributed to crystalline and amorphous sample regions, respectively. An increase of the sintering temperature leads to higher fractions of the fast lithium species with respect to the slow one, but hardly affects the jump dynamics in either of the phases. Specifically, the fast and slow lithium ions show jumps in the nanoseconds regime near 300 and 700 K, respectively. The activation energy of the hopping motion in the LATP crystallites amounts to ca. 0.26 eV. 7Li field-gradient diffusometry reveals that the long-range ion migration is limited by the sample regions featuring slow transport. The high spatial resolution available from the high static field gradients of our setup allows the observation of the lithium ion diffusion inside the small (D = 2 × 10−12 m2/s at room temperature.

We construct a segmental scale force level theory for the center-of-mass diffusion constant and corresponding relaxation time for globally compact unconcatenated ring polymer solutions and melts (degree of polymerization

Achieving mechanistic understanding of transport in complex environments such as inside cells or at polymer interfaces is challenging. We need better ways to image transport in 3-D and better single particle tracking algorithms to determine transport that are not systemically biased toward any classical motion model. Here we present an unbiased single particle tracking algorithm: Knowing Nothing Outside Tracking (KNOT). KNOT uses point clouds provided by iterative deconvolution to educate individual particle localizations and link particle positions between frames to achieve 2-D and 3-D tracking. Information from prior point clouds fuels an independent adaptive motion model for each particle to avoid global models that could introduce biases. KNOT competes with or surpasses other 2-D methods from the 2012 particle tracking challenge while accurately tracking adsorption dynamics of proteins on polymer surfaces and early endosome transport in live cells in 3-D. We apply KNOT to study 3-D endosome transport to reveal new physical insight into locally directed and diffusive transport in live cells. Our analysis demonstrates better accuracy in classifying local motion and its direction compared to previous methods, revealing intricate intracellular transport heterogeneities.

В работе исследуется нелинейная система обыкновенных дифференциальных уравнений, описывающая фотосинтез в автотрофных системах. Выделена область, инвариантная относительно движения вдоль траектории системы при возрастании времени. В этой области установлено существование единственного стационарного решения и исследованы вопросы его устойчивости. В настоящее время в связи с экспоненциальным ростом численности населения, индустриальным прогрессом и, как следствие, увеличением общего загрязнения биосферы, исследование устойчивости растительных организмов к антропогенным загрязнениям приобретают важнейшее практическое и теоретическое значение. Вместе с тем стала чрезвычайно актуальной проблема качественного исследования процессов фотосинтеза. В задачах, связанных с фотосинтезом, большой интерес представляет определение законов функционирования системы, а также выбор методов математического и компьютерного моделирования. Фотосинтез является значимым процессом нашей планеты. Это процесс преобразования поглощенной энергии света в химическую энергию органических соединений - единственный процесс в биосфере, ведущий к увеличению энергии биосферы за счёт внешнего источника - Солнца, обеспечивающего существование как растений, так и всех гетеротрофных организмов. Наиболее важными среди внешних факторов, влияющих на процессы фотосинтеза и фотодыхания, являются температура, фотосинтетически активная радиация, водный режим, режим минерального питания растения, а также содержание в окружающем пространстве углекислого газа и кислорода. В последние десятилетия наблюдается рост концентрации углекислого газа в атмосфере и изменение теплового режима в масштабах планеты. В связи с этим актуальной является задача прогнозирования изменения интенсивности фотосинтеза растений, обусловленного изменением концентрации атмосферного углекислого газа и температуры.

Hyaluronan is a biopolymer that is essential for many biological processes in the human body, like the regulation of tissue lubrication and inflammatory responses. Here, we study the behavior of hyaluronan at aqueous surfaces using heterodyne-detected vibrational sum-frequency generation spectroscopy (HD-VSFG). Low-molecular-weight hyaluronan (similar to 150 I(Da) gradually covers the water-air interface within hours, leading to a negatively charged surface and a reorientation of interfacial water molecules. The rate of surface accumulation strongly increases when the bulk concentration of low-molecular-weight hyaluronan is increased. In contrast, high-molecular-weight hyaluronan (>1 MDa) cannot be detected at the surface, even hours after the addition of the polymer to the aqueous solution. The strong dependence on the polymer molecular weight can be explained by entanglements of the hyaluronan polymers. We also find that for low-molecular-weight hyaluronan the migration kinetics of hyaluronan in aqueous media shows an anomalous dependence on the pH of the solution, which can be explained from the interplay of hydrogen bonding and electrostatic interactions of hyaluronan polymers.

The composition of cell-free expression systems (TX-TL) is adjusted by adding macromolecular crowding agents and salts. However, the effects of these cosolutes on the dynamics of individual gene expression processes have not been quantified. Here, we carry out kinetic mRNA and protein level measurements on libraries of genetic constructs using the common cosolutes PEG-8000, Ficoll-400, and magnesium glutamate. By combining these measurements with biophysical modeling, we show that cosolutes have differing effects on transcription initiation, translation initiation, and translation elongation rates with trade-offs between time delays, expression tunability, and maximum expression productivity. We also confirm that biophysical models can predict translation initiation rates in TX-TL using Escherichia coli lysate. We discuss how cosolute composition can be tuned to maximize performance across different cell-free applications, including biosensing, diagnostics, and biomanufacturing.

The effects, that glycine (Gly) has on the local chain conformation and internal backbone dynamics (IBD) of polypeptides, were investigated by comparing the fluorescence response of a series of pyr...

Na(+)/Ca(2+) exchangers (NCXs) are secondary active transporters that couple the translocation of Na(+) with the transport of Ca(2+) in the opposite direction. The exchanger is an essential Ca(2+) extrusion mechanism in excitable cells. It consists of a transmembrane domain and a large intracellular loop that contains two Ca(2+)-binding domains, CBD1 and CBD2. The two CBDs are adjacent to each other and form a two-domain Ca(2+) sensor called CBD12. Binding of intracellular Ca(2+) to CBD12 activates the NCX but inhibits the NCX of Drosophila, CALX. NMR spectroscopy and SAXS studies showed that CALX and NCX CBD12 constructs display significant interdomain flexibility in the apo state but assume rigid interdomain arrangements in the Ca(2+)-bound state. However, detailed structure information on CBD12 in the apo state is missing. Structural characterization of proteins formed by two or more domains connected by flexible linkers is notoriously challenging and requires the combination of orthogonal information from multiple sources. As an attempt to characterize the conformational ensemble of CALX-CBD12 in the apo state, we applied molecular dynamics (MD) simulations, NMR ((1)H-(15)N residual dipolar couplings), and small-angle x-ray scattering (SAXS) data in a combined strategy to select an ensemble of conformations in agreement with the experimental data. This joint approach demonstrated that CALX-CBD12 preferentially samples closed conformations, whereas the wide-open interdomain arrangement characteristic of the Ca(2+)-bound state is less frequently sampled. These results are consistent with the view that Ca(2+) binding shifts the CBD12 conformational ensemble toward extended conformers, which could be a key step in the NCXs’ allosteric regulation mechanism. This strategy, combining MD with NMR and SAXS, provides a powerful approach to select ensembles of conformations that could be applied to other flexible multidomain systems.

ispartof: JOURNAL OF THE CHINESE CHEMICAL SOCIETY vol:69 issue:1 pages:120-132 status: published

Lipid droplets (LDs) are dynamic lipid storage organelles needed for lipid homeostasis. Cells respond to metabolic changes by regulating the spatial distribution of LDs, as well as enzymes required for LD growth and turnover. Due to LD size below the optical diffraction limit, bulk fluorescence microscopy cannot observe the density and dynamics of specific LD enzymes. Here, we employ quantitative photo-activated localization microscopy (PALM) to study the density of the fatty acid activating protein Faa4 on LDs during log, stationary and lag phases in live yeast cells with single-molecule sensitivity and 30 nm resolution. During the log phase LDs co-localize with the Endoplasmic Reticulum (ER) where the highest Faa4 densities are measured. During transition to the stationary phase LDs translocate to the vacuolar surface and lumen with a ~2-fold increased surface area and a ~2.5-fold increase in Faa4 density, suggesting its role in LD expansion. The increased Faa4 density on LDs is caused by its ~5-fold increased expression level. When lipolysis is induced in stationary-phase cells by diluting them for 2 hrs in fresh medium, Faa4 shuttles to the vacuole through the two observed routes of ER- and lipophagy. The observed vacuolar localization of Faa4 may help activating fatty acids for membrane expansion and reduces Faa4 expression to levels found in the log phase.

Background: The human serotonin transporter is an important drug target for the treatment of various medical conditions of which depression is the most important, but also include attention deficit hyperactivity disorder, schizophrenia, social anxiety disorder and irritable bowel syndrome, among others. The transmembrane portion of the human transporter has been studied extensively and was first crystalized in 2016. However, the dynamical nature of the N-terminal segment of protein and its post-translational modifications remain insufficiently explored. Objective: The present study aims to evaluate the structure and dynamics of the N-terminal segment of the human serotonin transporter and the presence and stability of possible secondary structure elements along with its post-translational modifications and disorder propensity. Methods: The segment was investigated using a combination of bioinformatics tools for physicochemical characterization, secondary structure prediction, post-translational modifications and disorder prediction, followed by ab initio modeling and microsecond long explicit solvent molecular dynamics. Results: Our study reveals the presence of metastable secondary structure elements, namely two alpha helices and a beta-sheet, throughout the molecular dynamics run and identifies numerous sites with high probability for post-translational mod-ifications. Conclusion: Our results show that, despite the intrinsically unstructured nature, the N-terminus adopts a stable conformation with stable secondary structure elements, that could indicate an important functional role for the segment. Also, there is a high probability that the segment undergoes multiple post-translational modifications.

Conformational changes of antibodies and other biologics can decrease the effectiveness of pharmaceutical separations. Hence, a detailed mechanistic picture of antibody-stationary phase interactions that occur during ion-exchange chromatography (IEX) can provide critical insights. This work examines antibody conformational changes and how they perturb antibody motion and affect ensemble elution profiles. We combine IEX, three-dimensional single-protein tracking, and circular dichroism spectroscopy to investigate conformational changes of a model antibody, immunoglobulin G (IgG), as it interacts with the stationary phase as a function of salt conditions. The results indicate that the absence of salt enhances electrostatic attraction between IgG and the stationary phase, promotes surface-induced unfolding, slows IgG motion, and decreases elution from the column. Our results reveal previously unreported details of antibody structural changes and their influence on macroscale elution profiles.

Chemotactic migration of bacteria-their ability to direct multicellular motion along chemical gradients-is central to processes in agriculture, the environment, and medicine. However, current understanding of migration is based on studies performed in bulk liquid, despite the fact that many bacteria inhabit tight porous media such as soils, sediments, and biological gels. Here, we directly visualize the chemotactic migration of Escherichia coli populations in well-defined 3D porous media in the absence of any other imposed external forcing (e.g., flow). We find that pore-scale confinement is a strong regulator of migration. Strikingly, cells use a different primary mechanism to direct their motion in confinement than in bulk liquid. Furthermore, confinement markedly alters the dynamics and morphology of the migrating population-features that can be described by a continuum model, but only when standard motility parameters are substantially altered from their bulk liquid values to reflect the influence of pore-scale confinement. Our work thus provides a framework to predict and control the migration of bacteria, and active matter in general, in complex environments.

Phagocytosis is an important part of innate immunity and describes the engulfment of bacteria and other extracellular objects on the micrometer scale. The protrusion of the cell membrane around the bacteria during this process is driven by a reorganization of the actin cortex. The process has been studied on the molecular level to great extent during the past decades. However, a deep, fundamental understanding of the mechanics of the process is still lacking, in particular because of a lack of techniques that give access to binding dynamics below the optical resolution limit and cellular viscoelasticity at the same time. In this work, we propose a technique to characterize the mechanical properties of cells in a highly localized manner and apply it to investigate the early stages of phagocytosis. The technique can simultaneously resolve the contact region between a cell and an external object (in our application, a phagocytic target) even below the optical resolution limit. We used immunoglobulin-G-coated microparticles with a size of 2 μm as a model system and attached the particles to the macrophages with holographic optical tweezers. By switching the trap on and off, we were able to measure the rheological properties of the cells in a time-resolved manner during the first few minutes after attachment. The measured viscoelastic cellular response is consistent with power law rheology. The contact radius between particle and cell increased on a timescale of ∼30 s and converged after a few minutes. Although the binding dynamics are not affected by cytochalasin D, we observed an increase of the cellular compliance and a significant fluidization of the cortex after addition of cytochalasin D treatment. Furthermore, we report upper boundaries for the length- and timescale, at which cortical actin has been hypothesized to depolymerize during early phagocytosis.