Using High-Throughput Molecular Dynamics Simulation to Enhance the Computational Design of Kemp Elimination Enzymes.

Computational enzyme design has been successfully applied to identify new alternatives to natural enzymes for the biosynthesis of important compounds. However, the moderate catalytic activities of de novo designed enzymes indicate that the modeling accuracy of current computational enzyme design methods should be improved. Here, high-throughput molecular dynamics simulations were used to enhance computational enzyme design, thus allowing the identification of variants with higher activities in silico. Different time schemes of high-throughput molecular dynamics simulations were tested to identify the catalytic features of evolved Kemp eliminases. The 20 × 1 ns molecular dynamics simulation scheme was sufficiently accurate and computationally viable to screen the computationally designed massive variants of Kemp elimination enzymes. The developed hybrid computational strategy was used to redesign the most active Kemp eliminase, HG3.17, and five variants were generated and experimentally confirmed to afford higher catalytic efficiencies than that of HG3.17, with one double variant (D52Q/A53S) exhibiting a 55% increase. The hybrid computational enzyme design strategy is general and computationally economical, with which we anticipate the efficient creation of practical enzymes for industrial biocatalysis.

Transferability evaluation of the deep potential model for simulating water-graphene confined system.

Machine learning potentials (MLPs) are poised to combine the accuracy of ab initio predictions with the computational efficiency of classical molecular dynamics (MD) simulation. While great progress has been made over the last two decades in developing MLPs, there is still much to be done to evaluate their model transferability and facilitate their development. In this work, we construct two deep potential (DP) models for liquid water near graphene surfaces, Model S and Model F, with the latter having more training data. A concurrent learning algorithm (DP-GEN) is adopted to explore the configurational space beyond the scope of conventional ab initio MD simulation. By examining the performance of Model S, we find that an accurate prediction of atomic force does not imply an accurate prediction of system energy. The deviation from the relative atomic force alone is insufficient to assess the accuracy of the DP models. Based on the performance of Model F, we propose that the relative magnitude of the model deviation and the corresponding root-mean-square error of the original test dataset, including energy and atomic force, can serve as an indicator for evaluating the accuracy of the model prediction for a given structure, which is particularly applicable for large systems where density functional theory calculations are infeasible. In addition to the prediction accuracy of the model described above, we also briefly discuss simulation stability and its relationship to the former. Both are important aspects in assessing the transferability of the MLP model.

Importance of the Subunit-Subunit Interface in Ferritin Disassembly: A Molecular Dynamics Study.

Ferritin is a spherical cage-like protein that is useful for loading large functional particles for various applications. To our knowledge, how pH affects the interfaces inside ferritin and the mechanism of ferritin disassembly is far from complete. For this article, we conducted a series of molecular dynamics simulations (MD) at different pH values to study how interfaces affect ferritins' stability. It is shown that dimers are stable even at extremely low pH (pH 2.0), indicating that the dimer is the essential subunit for disassembly, and the slight swelling of the dimer resulting from monomer rotation inside a dimer is what triggers disassembly. During ferritin disassembly, there are two types of interfaces involved, and the interface between dimers is crucial. We also found that the driving forces for maintaining dimer stability are different when a dimer is inside ferritin and in an acidic solution. At low pH, the protonation of residues can lead to the loss of the salt bridge and the hydrogen bond between dimers, resulting in the disassembly of ferritin in an acidic environment. The above simulations reveal the possible mechanism of ferritin disassembly in an acidic solution, which can help us to design innovative and functional ferritin cages for different applications.

The role of conformational dynamics in the activity of polymer-conjugated CalB in organic solvents.

Perennial interest in enzyme catalysis has been expanding its applicability from aqueous phases where enzymes are naturally evolved to organic solvents in which the majority of industrial chemical syntheses are carried out. Although conjugating an enzyme with a soluble polymer has been attempted to enhance enzyme activity in organic solvents, the underlying mechanism remains poorly understood in terms of the conformational dynamics and enzyme activity. Herein, we combine LF-NMR measurements and MD simulations to investigate the effects of polymer grafting on the conformational dynamics of CalB in organic solvents and the consequential impacts on the catalytic kinetics, using the lipase-catalyzed transesterification reaction as a model system. LF-NMR measurements confirm that conjugation with a soluble polymer improves the enzyme flexibility in organic solvents, leading to an increase in the catalytic efficiency of up to two orders of magnitude. MD simulations suggest that the conjugated enzyme samples a larger conformational space, compared to its native counterpart, validating the hypothesis that polymer motion enhances enzyme dynamics. These experimental and simulation results provide new insights for enhancing enzyme conformational dynamics and thereby catalytic kinetics in organic solvents.

The synergistic mechanisms of apo-ferritin structural transitions and Au(iii) ion transportation: molecular dynamics simulations with the Markov state model.

Due to its unique structure, recent years have witnessed the use of apo-ferritin to accumulate various non-natural metal ions as a scaffold for nanomaterial synthesis. However, the transport mechanism of metal ions into the cavity of apo-ferritin is still unclear, limiting the rational design and controllable preparation of nanomaterials. Here, we conducted all-atom classical molecular dynamics (MD) simulations combined with Markov state models (MSMs) to explore the transportation behavior of Au(iii) ions. We exhibited the complete transportation paths of Au(iii) from solution into the apo-ferritin cage at the atomic level. We also revealed that the transportation of Au(iii) ions is accompanied by coupled protein structural changes. It is shown that the 3-fold axis channel serves as the only entrance with the longest residence time of Au(iii) ions. Besides, there are eight binding clusters and five 3-fold structural metastable states, which are important during Au(iii) transportation. The conformational changes of His118, Asp127, and Glu130, acting as doors, were observed to highly correlate with the Au(iii) ion's position. The MSM analysis and Potential Mean Force (PMF) calculation suggest a remarkable energy barrier near Glu130, making it the rate-limiting step of the whole process. The dominant transportation pathway is from cluster 3 in the 3-fold channel to the inner cavity to cluster 5 on the inner surface, and then to cluster 6. These findings provide inspiration and theoretical guidance for the further rational design and preparation of new nanomaterials using apo-ferritin.

A distal regulatory strategy of enzymes: from local to global conformational dynamics.

Modulating the distribution of various states in protein ensembles through distal sites may be promising in the evolution of enzymes in desired directions. However, the prediction of distal mutation hotspots that stabilize the favoured states from a computational perspective remains challenging. Here, we presented a strategy based on molecular dynamics (MD) and Markov state models (MSM) to predict distal mutation sites. Extensive MD combined with MSM was applied to determine the principally distributed metastable states interconverting at a slow timescale. Then, molecular docking was used to classify these states into active states and inactive ones. Distal mutation hotspots were targeted based on comparing the conformational features between active and inactive states, where mutations destabilize the inactive states and show little influence on the active state. The proposed strategy was used to explore the highly dynamic MHETase, which shows a potential application in the biodegradation of poly(ethylene terephthalate) (PET). Seven principally populated interrelated metastable states were identified, and the atomistic picture of their conformational changes was unveiled. Several residues at distal positions were found to adopt more H-bond occupancies in inactive states than active states, making them potential mutation hotspots for stabilizing the favoured conformations. In addition, the detailed mechanism revealed the significance of calcium ions at a distance from the catalytic centre in reshaping the free energy landscape. This study deepens the understanding of the conformational dynamics of α/ß hydrolases containing a lid domain and advances the study of enzymatic plastic degradation.

Ionic Transport Triggered by Asymmetric Illumination on 2D Nano-Membrane.

Ionic transport and ion sieving are important in the field of separation science and engineering. Based on the rapid development of nanomaterials and nano-devices, more and more phenomena occur on the nanoscale devices in the field of thermology, optics, mechanics, etc. Recently, we experimentally observed a novel ion transport phenomenon in nanostructured graphene oxide membrane (GOM) under asymmetric illumination. We first build a light-induced carriers' diffusion model based on our previous experimental results. This model can reveal the light-induced ion transport mechanism and predict the carriers' diffusion behavior under different operational situations and material characters. The voltage difference increases with the rise of illuminate asymmetry, photoresponsivity, recombination coefficient, and carriers' diffusion coefficient ratio. Finally, we discuss the ion transport behavior with different surface charge densities using MD simulation. Moderate surface charge decreases the ion transport with the same type of charge due to the electrostatic repulsion; however, excess surface charge blocks both cation and anion because a thicker electrical double layer decreases effective channel height. Research here provides referenced operational and material conditions to obtain a greater voltage difference between the membrane sides. Also, the mechanism of ion transport and ion sieving can guide us to modify membrane material according to different aims.

Light-Powered Directional Nanofluidic Ion Transport in Kirigami-Made Asymmetric Photonic-Ionic Devices.

Nacre-mimetic 2D nanofluidic materials with densely packed sub-nanometer-height lamellar channels find widespread applications in water-, energy-, and environment-related aspects by virtue of their scalable fabrication methods and exceptional transport properties. Recently, light-powered nanofluidic ion transport in synthetic materials gained considerable attention for its remote, noninvasive, and active control of the membrane transport property using the energy of light. Toward practical application, a critical challenge is to overcome the dependence on inhomogeneous or site-specific light illumination. Here, asymmetric photonic-ionic devices based on kirigami-tailored graphene oxide paper are fabricated, and directional nanofluidic ion transport properties therein powered by full-area light illumination are demonstrated. The in-plane asymmetry of the graphene oxide paper is essential to the generation of photoelectric driving force under homogeneous illumination. This light-powered ion transport phenomenon is explained based on a modified carrier diffusion model. In asymmetric nanofluidic structures, enhanced recombination of photoexcited charge carriers at the membrane boundary breaks the electric potential balance in the horizontal direction, and thus drives the ion transport in that direction under symmetric illumination. The kirigami-based strategy provides a facile and scalable way to fabricate paper-like photonic-ionic devices with arbitrary shapes, working as fundamental elements for large-scale light-harvesting nanofluidic circuits.

A hybrid theoretical method for predicting electrokinetic energy conversion in nanochannels.

The traditional methods to predict electrokinetic energy conversion (EKEC) in nanochannels are mostly based on the Navier-Stokes (NS) equation for ionic flow and the Poisson-Boltzmann (PB) equation for charge distributions, which is questionable for ion transport through highly charged nanochannels. In this work, the classical density functional theory (cDFT) is used together with molecular dynamics (MD) simulation and the Navier-Stokes (NS) equation to predict the electrical current and the thermodynamic efficiency of electrokinetic energy conversion in nanochannels. By introducing numerical results for the slip length calculated from MD simulation, a significant increase of the electrokinetic current is predicted in comparison to that obtained from the traditional electrokinetic equations with the non-slip boundary condition, leading to the theoretical predictions of the thermodynamic efficiency for electrokinetic energy conversion in nanochannels in good agreement with recent experiments. The hybrid method predicts that maximum electrokinetic efficiency can be achieved by tuning the channel height and solution conditions including electrolyte concentrations, ion valences, and surface energies. The theoretical results provide new insights into pressure-driven electrical energy generation processes and helpful guidelines for engineering design and optimization of electrokinetic energy conversion.

Single-molecule level dynamic observation of disassembly of the apo-ferritin cage in solution.

The ferritin cage iron-storage protein assembly has been widely used as a template for preparing nanomaterials. This assembly has a unique pH-induced disassembly/reassembly mechanism that provides a means for encapsulating molecules such as nanoparticles and small enzymes for catalytic and biomaterial applications. Although several researchers have investigated the disassembly process of ferritin, the dynamics involved in the initiation of the process and its intermediate states have not been elucidated due to a lack of suitable methodology to track the process in real-time. We describe the use of high-speed atomic force microscopy (HS-AFM) to image the dynamic event in real-time with single-molecule level resolution. The HS-AFM movies produced in the present work enable direct visualization of the movements of single ferritin cages in solution and formation of a hole prior to disassembly into subunit fragments. Additional support for these observations was confirmed at the atomic level by the results of all-atom molecular dynamics (MD) simulations, which revealed that the initiation process includes the opening of 3-fold symmetric channels. Our findings provide an essential contribution to a fundamental understanding of the dynamics of protein assembly and disassembly, as well as efforts to redesign the apo-ferritin cage for extended applications.

Highly Efficient Ionic Photocurrent Generation through WS2 -Based 2D Nanofluidic Channels.

The unique feature of nacre-like 2D layered materials provides a facile, yet highly efficient way to modulate the transmembrane ion transport from two orthogonal transport directions, either vertical or horizontal. Recently, light-driven active transport of ionic species in synthetic nanofluidic systems attracts broad research interest. Herein, taking advantage of the photoelectric semiconducting properties of 2D transition metal dichalcogenides, the generation of a directional and greatly enhanced cationic flow through WS2 -based 2D nanofluidic membranes upon asymmetric visible light illumination is reported. Compared with graphene-based materials, the magnitude of the ionic photocurrent can be enhanced by tens of times, and its photo-responsiveness can be 2-3.5 times faster. This enhancement is explained by the coexistence of semiconducting and metallic WS2 nanosheets in the hybrid membrane that facilitates the asymmetric diffusion of photoexcited charge carriers on the channel wall, and the high ionic conductance due to the neat membrane structure. To further demonstrate its application, photonic ion switches, photonic ion diodes, and photonic ion transistors as the fundamental elements for light-controlled nanofluidic circuits are further developed. Exploring new possibilities in the family of liquid processable colloidal 2D materials provides a way toward high-performance light-harvesting nanofluidic systems for artificial photosynthesis and sunlight-driven desalination.

Molecular dynamics simulations reveal how graphene oxide stabilizes and activates lipase in an anhydrous gas.

The interaction between Candida antarctica lipase B (CALB) and graphene oxide (GO) in an anhydrous gas was studied using molecular dynamics (MD) simulations augmented with a simulated annealing procedure to accelerate relaxation toward equilibrium. Three kinds of GO sheets with different oxygen contents were constructed to elucidate their effectiveness for stabilizing the active CALB conformation. It was shown that electrostatic forces are pivotal for the formation of CALB/GO complexes, and that a GO sheet with a higher oxygen content leads to stronger association with the protein. The simulation results suggest replacement of protein-binding water molecules by the GO surface, which was confirmed by thermogravimetric analysis. The CALB/GO assembly stabilizes the active enzyme conformation at elevated temperatures and, moreover, increases the protein flexibility near its active sites. The molecular details of GO interaction with CALB and the consequential effects on CALB stability and functionality are important for the development of unprecedented applications of gaseous enzymatic catalysis.

How Serratia marcescens HB-4 absorbs cadmium and its implication on phytoremediation.

A novel strain Serratia marcescens HB-4 with high Cadmium adsorption capacity was isolated from heavy metal contaminated soil in Hunan province, China. S. marcescens HB-4 reduced the concentration of Cd present in wastewater to less than 0.1 mg/L when the inlet stream contained no higher than 5.0 mg/L Cd. After treatment, wastewater meets Integrated Wastewater Discharge Standard of China (GB8978-1996). The naturally dead S. marcescens HB-4 still maintained over 80% of its Cd adsorption capacity. Scanning electron microscope (SEM), transmission electron microscope (TEM) and energy dispersive spectroscopy (EDS) results suggested that the mechanism of Cd adsorption can be explained as the synergy of extracellular adsorption, periplasm accumulation and intracellular absorption. The size of the accumulated Cd particular is at the nanometer scale, which can be washed out by EDTA without damaging cell integrity. SDS-polyacrylamide gel electrophoresis experiment showed that the heavy metal binding protein (especially Fe binding protein), transporter, amino acid and histidine periplasmic binding proteins and oxidoreductases were responsible for Cd removal. The pot experiment of S. marcescens HB-4 combined with Houttuynia cordata to detoxify Cd contaminated soil showed that the cadmium content in the aboveground and underground parts of Houttuynia cordata increased by 34.48% and 59.13% (w/w), respectively. The cadmium accumulation in Houttuynia cordata increased by 44.27% compared with the blank group which was not combined with S. marcescens HB-4. This work demonstrates that microbial synergistic phytoremediation has a significant potential to treat heavy metal contaminated soil.

In Situ Oxidation of Cu2O Crystal for Electrochemical Detection of Glucose.

The development of a sensitive, quick-responding, and robust glucose sensor is consistently pursued for use in numerous applications. Here, we propose a new method for preparing a Cu2O electrode for the electrochemical detection of glucose concentration. The Cu2O glucose electrode was prepared by in situ electrical oxidation in an alkaline solution, in which Cu2O nanoparticles were deposited on the electrode surface to form a thin film, followed by the growth of Cu(OH)2 nanorods or nanotubes. The morphology and electrocatalytic activity of a Cu2O glucose electrode can be tuned by the current density, reaction time, and NaOH concentration. The results from XRD, SEM, and a Raman spectrum show that the electrode surface was coated with cubic Cu2O nanoparticles with diameters ranging from 50 to 150 nm. The electrode exhibited a detection limit of 0.0275 mM, a peak sensitivity of 2524.9 µA·cm-2·mM-1, and a linear response range from 0.1 to 1 mM. The presence of high concentrations of ascorbic acid, uric acid, dopamine and lactose appeared to have no effects on the detection of glucose, indicating a high specificity and robustness of this electrode.

A molecular theory for predicting the thermodynamic efficiency of electrokinetic energy conversion in slit nanochannels.

The classical density functional theory is incorporated with the Stokes equation to examine the thermodynamic efficiency of pressure-driven electrokinetic energy conversion in slit nanochannels. Different from previous mean-field predictions, but in good agreement with recent experiments, the molecular theory indicates that the thermodynamic efficiency may not be linearly correlated with the channel size or the electrolyte concentration. For a given electrolyte, an optimal slit nanochannel size and ion concentration can be identified to maximize both the electrical current and the thermodynamic efficiency. The optimal conditions are sensitive to a large number of parameters including ion diameters, valences, electrolyte concentration, channel size, and the valence- and size-asymmetry of oppositely charged ionic species. The theoretical results offer fresh insights into pressure-driven current generation processes and are helpful guidelines for the design of apparatus for the electrokinetic energy conversion.

Markov-state model for CO2 binding with carbonic anhydrase under confinement.

Enzyme immobilization with a nanostructure material can enhance its stability and facilitate reusability. However, the apparent activity is often compromised due to additional diffusion barriers and complex interactions with the substrates and solvent molecules. The present study elucidates the effects of the surface hydrophobicity of nano-confinement on CO2 diffusion to the active site of human carbonic anhydrase II (CA), an enzyme that is able to catalyze CO2 hydration at extremely high turnover rates. Using the Markov-state model in combination with coarse-grained molecular dynamics simulations, we demonstrate that a hydrophobic cage increases CO2 local density but hinders its diffusion towards the active site of CA under confinement. By contrast, a hydrophilic cage hinders CO2 adsorption but promotes its binding with CA. An optimal surface hydrophobicity can be identified to maximize both the CO2 occupation probability and the diffusion rate. The simulation results offer insight into understanding enzyme performance under nano-confinement and help us to advance broader applications of CA for CO2 absorption and recovery.

The Mechanism for siRNA Transmembrane Assisted by PMAL.

The capacity of silencing genes makes small interfering RNA (siRNA) appealing for curing fatal diseases. However, the naked siRNA is vulnerable to and degraded by endogenous enzymes and is too large and too negatively charged to cross cellular membranes. An effective siRNA carrier, PMAL (poly(maleic anhydride-alt-1-decene) substituted with 3-(dimethylamino) propylamine), has been demonstrated to be able to assist siRNA transmembrane by both experiments and molecular simulation. In the present work, the mechanism of siRNA transmembrane assisted by PMAL was studied using steered molecular dynamics simulations based on the martini coarse-grained model. Here two pulling rates, i.e., 10−6 and 10−5 nm·ps−1, were chosen to imitate the passive and active transport of siRNA, respectively. Potential of mean force (PMF) and interactions among siRNA, PMAL, and lipid bilayer membrane were calculated to describe the energy change during siRNA transmembrane processes at various conditions. It is shown that PMAL-assisted siRNA delivery is in the mode of passive transport. The PMAL can help siRNA insert into lipid bilayer membrane by lowering the energy barrier caused by siRNA and lipid bilayer membrane. PMAL prefers to remain in the lipid bilayer membrane and release siRNA. The above simulations establish a molecular insight of the interaction between siRNA and PMAL and are helpful for the design and applications of new carriers for siRNA delivery.

Kinetics of CO2 diffusion in human carbonic anhydrase: a study using molecular dynamics simulations and the Markov-state model.

Molecular dynamics (MD) simulations, in combination with the Markov-state model (MSM), were applied to probe CO2 diffusion from an aqueous solution into the active site of human carbonic anhydrase II (hCA-II), an enzyme useful for enhanced CO2 capture and utilization. The diffusion process in the hydrophobic pocket of hCA-II was illustrated in terms of a two-dimensional free-energy landscape. We found that CO2 diffusion in hCA-II is a rate-limiting step in the CO2 diffusion-binding-reaction process. The equilibrium distribution of CO2 shows its preferential accumulation within a hydrophobic domain in the protein core region. An analysis of the committors and reactive fluxes indicates that the main pathway for CO2 diffusion into the active site of hCA-II is through a binding pocket where residue Gln136 contributes to the maximal flux. The simulation results offer a new perspective on the CO2 hydration kinetics and useful insights toward the development of novel biochemical processes for more efficient CO2 sequestration and utilization.

A theoretical study on the morphological phase diagram of supported lipid bilayers.

Supported lipid bilayers (SLBs) have been widely used in drug delivery, biosensors and biomimetic membranes. The microscopic mechanism of SLB formation and stability depends on a number of factors underlying solvent-mediated lipid-lipid and lipid-substrate interactions. Whereas recent years have witnessed remarkable progress in understanding the kinetics of SLB formation, relatively little is known about the lipid phase behavior controlling the SLB stability under diverse solution conditions. In this work, we examine the structure of SLBs using classical density functional theory (CDFT) in the context of a coarse-grained model that accounts for ion-explicit electrostatic interactions, surface hydrophobicity, as well as the molecular characteristics of the lipid tails. A morphological phase diagram is constructed in terms of various intrinsic properties of lipid molecules (such as the lipid tail length, size and charge of the lipid head segments), substrate conditions (such as the surface charge density and hydrophobicity), and solution parameters (such as the ion concentration and ion type). The morphological phase diagram provides useful insights into the rational design and broader application of SLB membranes as different types of nano-devices.

Spreading of a Unilamellar Liposome on Charged Substrates: A Coarse-Grained Molecular Simulation.

Supported lipid bilayers (SLBs) are able to accommodate membrane proteins useful for diverse biomimetic applications. Although liposome spreading represents a common procedure for preparation of SLBs, the underlying mechanism is not yet fully understood, particularly from a molecular perspective. The present study examines the effects of the substrate charge on unilamellar liposome spreading on the basis of molecular dynamics simulations for a coarse-grained model of the solvent and lipid molecules. Liposome transformation into a lipid bilayer of different microscopic structures suggests three types of kinetic pathways depending on the substrate charge density, that is, top-receding, parachute, and parachute with wormholes. Each pathway leads to a unique distribution of the lipid molecules and thereby distinctive properties of SLBs. An increase of the substrate charge density results in a magnified asymmetry of the SLBs in terms of the ratio of charged lipids, parallel surface movements, and the distribution of lipid molecules. While the lipid mobility in the proximal layer is strongly correlated with the substrate potential, the dynamics of lipid molecules in the distal monolayer is similar to that of a freestanding lipid bilayer. For liposome spreading on a highly charged surface, wormhole formation promotes lipid exchange between the SLB monolayers thus reduces the asymmetry on the number density of lipid molecules, the lipid order parameter, and the monolayer thickness. The simulation results reveal the important regulatory role of electrostatic interactions on liposome spreading and the properties of SLBs.