Construction of T‐Motif‐Based DNA Nanostructures through Enzymatic Reactions

The most common way to fabricate DNA nanostructures is to mix individually synthesized DNA oligomers in one pot. However, if DNA nanostructures could be produced through enzymatic reactions, they could be applied in various environments, including in vivo. Herein, an enzymatic method developed to construct a DNA nanostructure from a simple motif called a T‐motif is reported. A long, repeated structure was replicated from a circular template by rolling circle amplification and then cleaved into T‐motif segments by restriction enzymes. These motifs have been successfully assembled into a ladder‐like nanostructure without purification or controlled annealing. This approach is widely applicable to constructing a variety of DNA nanostructures through enzymatic reactions.     

Role of Monovalent Ions in the NKCC1 Inhibition Mechanism Revealed through Molecular Simulations

The secondary active Na-K-Cl cotransporter 1 (NKCC1) promotes electroneutral uptake of two chloride ions, one sodium ion and one potassium ion. NKCC1 regulates Cl⁻ homeostasis, thus being implicated in transepithelial water transport and in neuronal excitability. Aberrant NKCC1 transport is linked to a variety of human diseases. The loop diuretic drugs bumetanide, furosemide, azosemide and ethacrynic acid target NKCC1, but are characterized by poor selectivity leading to severe side effects. Despite its therapeutic importance, the molecular details of the NKCC1 inhibition mechanism remain unclear. Using all-atom simulations, we predict a putative binding mode of these drugs to the zebrafish (z) and human (h) NKCC1 orthologs. Although differing in their specific interactions with NKCC1 and/or monovalent ions, all drugs can fit within the same cavity and engage in hydrophobic interactions with M304/M382 in z/hNKCC1, a proposed ion gating residue demonstrated to be key for bumetanide binding. Consistent with experimental evidence, all drugs take advantage of the K⁺/Na⁺ ions, which plastically respond to their binding. This study not only provides atomic-level insights useful for drug discovery campaigns of more selective/potent NKCC1 inhibitors aimed to tackle diseases related to deregulated Cl⁻ homeostasis, but it also supplies a paradigmatic example of the key importance of dynamical effects when drug binding is mediated by monovalent ions.     

Base Flip in DNA Studied by Molecular Dynamics Simulations of Differently-Oxidized Forms of Methyl-Cytosine

Distortions in the DNA sequence, such as damage or mispairs, are specifically recognized and processed by DNA repair enzymes. Many repair proteins and, in particular, glycosylases flip the target base out of the DNA helix into the enzyme’s active site. Our molecular dynamics simulations of DNA with intact and damaged (oxidized) methyl-cytosine show that the probability of being flipped is similar for damaged and intact methyl-cytosine. However, the accessibility of the different 5-methyl groups allows direct discrimination of the oxidized forms. Hydrogen-bonded patterns that vary between methyl-cytosine forms carrying a carbonyl oxygen atom are likely to be detected by the repair enzymes and may thus help target site recognition.     

Numerical study on electrophoretical stretching dynamics of DNA in microcontraction

Brownian dynamics simulations are used to characterize the electrophoretical stretching process of long T4 DNA in microchannels. When DNA is forced to move through the microchannels, the pure elongational flow generated by electric field gradients in hyperbolic contraction will unravel the molecules of DNA. The effects of hydrodynamic interactions, the strain rate, the Brownian fluctuation, and the initial states of molecules on the stretching dynamics are analyzed in this paper. The computational results show us the weak dependence of polymer dynamics on hydrodynamic interactions in microcontractions. In the case of low Deborah number, the stretching process of a molecule depends on the Brownian fluctuation. However, in the case of high Deborah number, the individualistic stretching behavior can be traced to variations in the starting conformation.     

Electrophoretical stretching of DNA in a hybrid microchannel

The electrophoretical stretching of DNA in a hybrid microchannel is analyzed in this paper. The channel comprises a large insulating cylinder and a hyperbolic contraction that can cause the DNA deformation. Brownian dynamics simulation is used to characterize the dynamical stretching process of a long T4 DNA in hybrid microchannels. The computational results show us the larger average extension of DNA in cylinder-hybridized microchannels than that in the single microcontractions due to the prestretched effect of cylinder on DNA. Moreover, the location and the radius of the insulating cylinder in hybrid microchannels have great effects on the stretching behavior of DNA.     

Artificial Metalloenzymes for Asymmetric Catalysis by Creation of Novel Active Sites in Protein and DNA Scaffolds

Artificial metalloenzymes have emerged as a promising new approach to asymmetric catalysis. In our group, we are exploring novel artificial metalloenzyme designs involving creation of a new active site in a protein or DNA scaffold that does not have an existing binding pocket. In this review, we give an overview of the developments in the two approaches to artificial metalloenzymes for asymmetric catalysis investigated in our group: creation of a novel active site on a peptide or protein dimer interface and using DNA as a scaffold for artificial metalloenzymes.     

Molecular Modeling and Molecular Dynamics Studies of Hydralazine with Human DNA Methyltransferase 1

A series of DNA methyltransferase 1 (DNMT1) inhibitors were modeled by docking and molecular dynamics studies to rationalize their activity. Our findings will be valuable in guiding research efforts toward the rational design and virtual screening of novel DNMT inhibitors.

     

Universal Properties and Specificities of the β2-Adrenergic Receptor-Gs Protein Complex Activation Mechanism Revealed by All-Atom Molecular Dynamics Simulations

G protein-coupled receptors (GPCRs) are transmembrane proteins of high pharmacological relevance. It has been proposed that their activity is linked to structurally distinct, dynamically interconverting functional states and the process of activation relies on an interconnecting network of conformational switches in the transmembrane domain. However, it is yet to be uncovered how ligands with different extents of functional effect exert their actions. According to our recent hypothesis, based on indirect observations and the literature data, the transmission of the external stimulus to the intracellular surface is accompanied by the shift of macroscopic polarization in the transmembrane domain, furnished by concerted movements of highly conserved polar motifs and the rearrangement of polar species. In this follow-up study, we have examined the β₂-adrenergic receptor (β₂AR) to see if our hypothesis drawn from an extensive study of the μ-opioid receptor (MOP) is fundamental and directly transferable to other class A GPCRs. We have found that there are some general similarities between the two receptors, in agreement with previous studies, and there are some receptor-specific differences that could be associated with different signaling pathways.     

Stereospecific Effects of Oxygen‐to‐Sulfur Substitution in DNA Phosphate on Ion Pair Dynamics and Protein–DNA Affinity

Oxygen‐to‐sulfur substitutions in DNA phosphate often enhance affinity for DNA‐binding proteins. Our previous studies have suggested that this effect of sulfur substitution of both O_P1 and O_P2 atoms is due to an entropic gain associated with enhanced ion pair dynamics. In this work, we studied stereospecific effects of single sulfur substitution of either the O_P1 or O_P2 atom in DNA phosphate at the Lys57 interaction site of the Antennapedia homeodomain–DNA complex. Using crystallography, we obtained structural information on the R_P and S_P diastereomers of the phosphoromonothioate and their interaction with Lys57. Using fluorescence‐based assays, we found significant affinity enhancement upon sulfur substitution of the O_P2 atom. Using NMR spectroscopy, we found significant mobilization of the Lys57 side‐chain NH₃⁺ group upon sulfur substitution of the O_P2 atom. These data provide further mechanistic insights into the affinity enhancement by oxygen‐to‐sulfur substitution in DNA phosphate.     

Asymmetric Synthesis of Substituted Thiolanes through Domino Thia‐Michael–Henry Dynamic Covalent Systemic Resolution using Lipase Catalysis

Dynamic systems based on consecutive thia‐Michael and Henry reactions were generated and transformed using lipase‐catalyzed asymmetric transformation. Substituted thiolane structures with three contiguous stereocenters were resolved in the process in high yields and high enantiomeric excesses.

     

Discovery of Highly Potent CRBN Ligands and Insight into Their Binding Mode through Molecular Docking and Molecular Dynamics Simulations

Cereblon (CRBN) is a substrate receptor of E3 ubiquitin ligase as well as the target of thalidomide and lenalidomide, plays a vital role in endogenous protein degradation. In this article, two series of compounds with novel structure were designed, synthesized and evaluated against CRBN. YJ1b, designed based on our previous finding, shown strong binding affinity toward CRBN (IC₅₀=0.206 μM) by forming a salt bridge interaction with amino acid residue Glu377 of CRBN, it was 13-fold compared with that of lenalidomide (IC₅₀=2.694 μM) in TR-FRET assay. YJ2c and YJ2h, two analogs of YJ1b, also exhibit high binding affinity toward CRBN (IC₅₀=0.211 μM and IC₅₀=0.282 μM, respectively). While, molecular docking and 100 ns molecular dynamic simulation studies were conducted to insight into the unique binding mode of YJ1b, YJ2c and YJ2e toward CRBN. The new compounds with special binding mode in this article may serve for the further optimization and discovery of novel high potent CRBN ligands.     

Simulations of scalar dispersion in fluidized solid–liquid suspensions

Direct, particle‐resolved simulations of solid–liquid fluidization with the aim of quantifying dispersion have been performed. In addition to simulating the multiphase flow dynamics (that is dealt with by a lattice‐Boltzmann method coupled to an event‐driven hard‐sphere algorithm), a transport equation of a passive scalar in the liquid phase has been solved by means of a finite‐volume approach. The spreading of the scalar—as a consequence of the motion of the fluidized, monosized spherical particles that agitate the liquid—is quantified through dispersion coefficients. Particle self‐diffusivities have also been determined. Solids volume fractions were in the range 0.2–0.5, whereas single‐sphere settling Reynolds numbers varied between approximately 3 and 20. The dispersion processes are highly anisotropic with lateral spreading much slower (by one order of magnitude) than vertical spreading. Scalar dispersion coefficients are of the same order of magnitude as particle self‐diffusivities. © 2014 American Institute of Chemical Engineers AIChE J, 60: 1880–1890, 2014     

Application of Molecular Dynamics Simulations in the Analysis of Cyclodextrin Complexes

Cyclodextrins (CDs) are highly respected for their ability to form inclusion complexes via host–guest noncovalent interactions and, thus, ensofance other molecular properties. Various molecular modeling methods have found their applications in the analysis of those complexes. However, as showed in this review, molecular dynamics (MD) simulations could provide the information unobtainable by any other means. It is therefore not surprising that published works on MD simulations used in this field have rapidly increased since the early 2010s. This review provides an overview of the successful applications of MD simulations in the studies on CD complexes. Information that is crucial for MD simulations, such as application of force fields, the length of the simulation, or solvent treatment method, are thoroughly discussed. Therefore, this work can serve as a guide to properly set up such calculations and analyze their results.     

Single‐Molecule Unzipping Force Analysis of HU–DNA Complexes

The genome of bacteria is organized and compacted by the action of nucleoid‐associated proteins. These proteins are often present in tens of thousands of copies and bind with low specificity along the genome. DNA‐bound proteins thus potentially act as roadblocks to the progression of machinery that moves along the DNA. In this study, we have investigated the effect of histone‐like protein from strain U93 (HU), one of the key proteins involved in shaping the bacterial nucleoid, on DNA helix stability by mechanically unzipping single dsDNA molecules. Our study demonstrates that individually bound HU proteins have no observable effect on DNA helix stability, whereas HU proteins bound side‐by‐side within filaments increase DNA helix stability. As the stabilizing effect is small compared to the power of DNA‐based motor enzymes, our results suggest that HU alone does not provide substantial hindrance to the motor's progression in vivo.     

The role of DNA in PANI–DNA hybrid: Template and dopant

We exposed a novel method by using DNA as the dopant as well as template at the same time to prepare PANI–DNA hybrid micro/nanowires with conductivity as high as 10⁻² S cm⁻¹. The high conductivity is due to the co-doping function of DNA with HCl produced by FeCl₃. It is found that the morphology and conductivity of the PANI–DNA hybrids are affected by the [DNA]/[AN] ratio due to the co-operation and competition of DAN's dopant and template function, and the role of DNA in PANI–DNA hybrid varies with the changing of [DNA/[AN] ratios.     

Proteomic Analysis Revealed Different Molecular Mechanisms of Response to PEG Stress in Drought-Sensitive and Drought-Resistant Sorghums

Drought is the major limiting factor that directly or indirectly inhibits the growth and reduces the productivity of sorghum (Sorghum bicolor (L.) Moench). As the main vegetative organ of sorghum, the response mechanism of the leaf to drought stress at the proteomic level has not been clarified. In the present study, nano-scale liquid chromatography mass spectrometry (nano-LC-MS/MS) technology was used to compare the changes in the protein expression profile of the leaves of drought-sensitive (S4 and S4-1) and drought-resistant (T33 and T14) sorghum varieties at the seedling stage under 25% PEG-6000 treatment for 24 h. A total of 3927 proteins were accurately quantitated and 46, 36, 35, and 102 differentially abundant proteins (DAPs) were obtained in the S4, S4-1, T14, and T33 varieties, respectively. Four proteins were randomly selected for parallel reaction monitoring (PRM) assays, and the results verified the reliability of the mass spectrometry (MS) results. The response mechanism of the drought-sensitive sorghum leaves to drought was attributed to the upregulation of proteins involved in the tyrosine metabolism pathway with defense functions. Drought-resistant sorghum leaves respond to drought by promoting the TCA cycle, enhancing sphingolipid biosynthesis, interfering with triterpenoid metabolite synthesis, and influencing aminoacyl-tRNA biosynthesis. The 17 screened important candidate proteins related to drought stress were verified by quantitative real-time PCR (qRT-PCR), the results of which were consistent with the results of the proteomic analysis. This study lays the foundation for revealing the drought-resistance mechanism of sorghum at the protein level. These findings will help us cultivate and improve new drought-resistant sorghum varieties.     

Signaling Role of Mitochondrial Enzymes and Ultrastructure in the Formation of Molecular Mechanisms of Adaptation to Hypoxia

This study was the first comprehensive investigation of the dependence of mitochondrial enzyme response (catalytic subunits of mitochondrial complexes (MC) I-V, including NDUFV2, SDHA, Cyt b, COX1 and ATP5A) and mitochondrial ultrastructure in the rat cerebral cortex (CC) on the severity and duration of in vivo hypoxic exposures. The role of individual animal’s resistance to hypoxia was also studied. The respiratory chain (RC) was shown to respond to changes in environmental [O₂] as follows: (a) differential reaction of mitochondrial enzymes, which depends on the severity of the hypoxic exposure and which indicates changes in the content and catalytic properties of mitochondrial enzymes, both during acute and multiple exposures; and (b) ultrastructural changes in mitochondria, which reflect various degrees of mitochondrial energization. Within a specific range of reduced O₂ concentrations, activation of the MC II is a compensatory response supporting the RC electron transport function. In this process, MC I develops new kinetic properties, and its function recovers in hypoxia by reprograming the RC substrate site. Therefore, the mitochondrial RC performs as an in vivo molecular oxygen sensor. Substantial differences between responses of rats with high and low resistance to hypoxia were determined.     

Structure and Thermal Stability of wtRop and RM6 Proteins through All-Atom Molecular Dynamics Simulations and Experiments

In the current work we study, via molecular simulations and experiments, the folding and stability of proteins from the tertiary motif of 4-α-helical bundles, a recurrent motif consisting of four amphipathic α-helices packed in a parallel or antiparallel fashion. The focus is on the role of the loop region in the structure and the properties of the wild-type Rop (wtRop) and RM6 proteins, exploring the key factors which can affect them, through all-atom molecular dynamics (MD) simulations and supporting by experimental findings. A detailed investigation of structural and conformational properties of wtRop and its RM6 loopless mutation is presented, which display different physical characteristics even in their native states. Then, the thermal stability of both proteins is explored showing RM6 as more thermostable than wtRop through all studied measures. Deviations from native structures are detected mostly in tails and loop regions and most flexible residues are indicated. Decrease of hydrogen bonds with the increase of temperature is observed, as well as reduction of hydrophobic contacts in both proteins. Experimental data from circular dichroism spectroscopy (CD), are also presented, highlighting the effect of temperature on the structural integrity of wtRop and RM6. The central goal of this study is to explore on the atomic level how a protein mutation can cause major changes in its physical properties, like its structural stability.