Chemical interference with DSIF complex formation lowers synthesis of mutant huntingtin gene products and curtails mutant phenotypes.

Earlier work has shown that siRNA-mediated reduction of the SUPT4H or SUPT5H proteins, which interact to form the DSIF complex and facilitate transcript elongation by RNA polymerase II (RNAPII), can decrease expression of mutant gene alleles containing nucleotide repeat expansions differentially. Using luminescence and fluorescence assays, we identified chemical compounds that interfere with the SUPT4H-SUPT5H interaction and then investigated their effects on synthesis of mRNA and protein encoded by mutant alleles containing repeat expansions in the huntingtin gene (HTT), which causes the inherited neurodegenerative disorder, Huntington's Disease (HD). Here we report that such chemical interference can differentially affect expression of HTT mutant alleles, and that a prototypical chemical, 6-azauridine (6-AZA), that targets the SUPT4H-SUPT5H interaction can modify the biological response to mutant HTT gene expression. Selective and dose-dependent effects of 6-AZA on expression of HTT alleles containing nucleotide repeat expansions were seen in multiple types of cells cultured in vitro, and in a Drosophila melanogaster animal model for HD. Lowering of mutant HD protein and mitigation of the Drosophila "rough eye" phenotype associated with degeneration of photoreceptor neurons in vivo were observed. Our findings indicate that chemical interference with DSIF complex formation can decrease biochemical and phenotypic effects of nucleotide repeat expansions.

Molecular Dynamics Simulations of High-Performance, Dissipationless Desalination across Self-Assembled Amyloid Beta Nanotubes.

Climate change is causing droughts and water shortages. Membrane desalination is one of the most widely employed conventional methods of creating a source of clean water, but is a very energy-intensive process. Membrane separation requires high salt selectivity across nano-channels, yet traditional techniques remain inefficient in this regard. Herein, a bioinspired, chemically robust, amyloid-fibril-based nanotube is designed, exhibiting water permeability and salt rejection properties capable of providing highly efficient desalination. Molecular dynamics simulations show that nano-dewetting facilitates the unidirectional motion of water molecules on the surface of amyloid beta (Aß) sheets owing to the ratchet structure of the underlying potential surface and the broken detailed balance. The water inside the self-assembled Aß nanotube (ABNT) overflows, while the passage of salts can be blocked using amphiphilic peptides. The designed nanofilter ABNT shows 100% desalination efficiency with perfect NaCl rejection. The production of ≈2.5 tons of pure water per day without any energy input, which corresponds to a water flux up to 200 times higher than those of existing commercial methods, is assessed by this simulation method. These results provide a detailed fundamental understanding of potential high-performance nanotechnologies for water treatment.

Interfacial water effect on cooperativity and signal communication in Scapharca dimeric hemoglobin.

Cooperativity is important in controlling the biological functions of allosteric proteins. Understanding the detailed mechanisms of cooperativity and allosteric regulation in such proteins is essential to understanding their function; however, the mechanism by which allosteric proteins undergo conformational transitions to aid the ligand escape process and its relevance to interfacial water molecules is not well understood. Here, we perform molecular dynamics simulations to examine these issues in Scapharca dimeric hemoglobin. The effects of interfacial water on dimeric motion, ligand escape probability, gate function, and cross-correlation are considered. The results reveal that interfacial water exhibits an unbalanced stress distribution in the interface region, leading to a bias helix bundle motion that not only can expedite the escape of the first ligand but also can increase the interval between the escape of both ligands. Correspondingly, the gate function follows the same time scale as the F-helix movement, and the gate opening is non-stochastic; moreover, the inconsistent motion between the gate parts resembles cooperative behavior. An explicit analysis of the intersubunit communication map provides at least 14 signal transduction pathways. Our results significantly aid in understanding the role of interfacial water in manipulating cooperativity and will lead to further applications involving molecular machines.

Directed motion from particle size oscillations inside an asymmetric channel.

The motion of a spherical Brownian particle in an asymmetric periodic channel is considered. Under an external periodic stimulus, the particle switches between two states with different particle radius, every half-period. Using Brownian dynamics simulations, we show that the particle size oscillation, combined with the asymmetry of the channel, induces a drift along the channel axis, directed towards the steeper wall of the channel. The oscillation of the particle size is accompanied by a time variation of the space accessible to the particle and by an oscillation of its diffusion coefficient. The former underlies the drift inducing mechanism of purely entropic nature. The latter, combined with the former, leads to a significant amplification of the effect. The drift velocity vanishes when interconversion between the states occurs either very slow or very fast, having a maximum in between. The position and magnitude of the maximum are discussed by providing an analytical approach based on intuitively appealing assumptions.

A model for ultra-fast charge transport in membrane proteins.

Isolated proteins have recently been observed to transport charge and reactivity over very long distances with extraordinary rates and near perfect efficiencies in spite of their site. This is not the case if the peptide is in water, where the efficiency of charge hopping to the next site is reduced to approximately 2%. Here, water is not an ideal solvent for charge transport. The issue at hand is how to explain such enormous charge transfer quenching in water compared to another typical medium, namely lipid. We performed molecular dynamics simulations to computationally substantiate the novel long-distance charge transfer yield of the polypeptides in lipids. This is characterized by the charge transfer persistent-distance decay constant and not by the rate, which is seldom, if ever, measured and hence not directly addressed here. This model can encompass an extremely wide range of yields over very long distances in peptides in various media. The calculations here demonstrate the good charge transport efficiency in lipids in contrast to the poor efficiency in water. The protein charge transport also exhibits a very strong anisotropic effect in lipids. The peptide secondary structure effect of charge transfer in membranes is analyzed in contrast to that in water. These results suggest that this model can be useful for the prediction of charge transfer efficiency in various environments of interest and indicate that the charge transfer is highly efficient in membrane proteins.

The molecular mechanism of ligand unbinding from the human telomeric G-quadruplex by steered molecular dynamics and umbrella sampling simulations.

G-quadruplexes are attractive drug targets in cancer therapy. Understanding the mechanisms of the binding-unbinding processes involving biomolecules and molecular recognition is essential for designing new drugs of G-quadruplexes. We performed steered molecular dynamics and umbrella sampling simulations to investigate the molecular mechanism and kinetics of ligand unbinding processes of the basket, propeller and hybrid G-quadruplex structures. Our studies of the ligand charge effect showed that Coulomb interaction plays a significant role in stabilizing the G-quadruplex structure in the unbinding process. The free energy profiles were carried out and the free energy changes associated with the unbinding process were computed quantitatively, whereas these results could help to identify accessible binding sites and transient interactions. The dynamics of the hydration shell water molecules around the G-quadruplex exhibits an abnormal Brownian motion, and the thickness and free energy of the hydration shell were estimated. A two-step relaxation scheme was theoretically developed to describe the kinetic reaction of BMVC and G-quadruplex interactions. Our computed results fall in a reasonable range of experimental data. The present investigation could be helpful in the structure-based drug design.

Relative stability of G-quadruplex structures: Interactions between the human Bcl2 promoter region and derivatives of carbazole and diphenylamine.

The bcl2 promoter region forms a G-quadruplex structure, which is a crucial target for anticancer drug development. In this study, we provide theoretical predictions of the stability of different G-quadruplex folds of the 23-mer bcl2 promoter region and G-quadruplex ligand. We take into account the whole G-quadruplex structure, including bound-cations and solvent effects, in order to compute the ligand binding free energy using molecular dynamics simulation. Two series of the carbazole and diphenylamine derivatives are used to screen for the most potent drug in terms of stabilization. The energy analysis identifies the predominant energy components affecting the stability of the various different G-quadruplex folds. The energy associated with the stability of the G-quadruplex-K(+) structures obtained displays good correlation with experimental Tm measurements. We found that loop orientation has an intrinsic influence on G-quadruplex stability and that the basket structure is the most stable. Furthermore, parallel loops are the most effective drug binding site. Our studies also demonstrate that rigidity and planarity are the key structural elements of a drug that stabilizes the G-quadruplex structure. BMVC-4 is the most potential G-quadruplex ligand. This approach demonstrates significant promise and should benefit drug design.

Fluctuation-induced transport of two coupled particles: effect of the interparticle interaction.

We consider a system of two coupled particles fluctuating between two states, with different interparticle interaction potentials and particle friction coefficients. An external action drives the interstate transitions that induces reciprocating motion along the internal coordinate x (the interparticle distance). The system moves unidirectionally due to rectification of the internal motion by asymmetric friction fluctuations and thus operates as a dimeric motor that converts input energy into net movement. We focus on how the law of interaction between the particles affects the dimer transport and, in particular, the role of thermal noise in the motion inducing mechanism. It is argued that if the interaction potential behaves at large distances as x(α), depending on the value of the exponent α, the thermal noise plays a constructive (α > 2), neutral (α = 2), or destructive (α < 2) role. In the case of α = 1, corresponding piecewise linear potential profiles, an exact solution is obtained and discussed in detail.

Structural insights into the catalytic active site and activity of human Nit2/ω-amidase: kinetic assay and molecular dynamics simulation.

Human nitrilase-like protein 2 (hNit2) is a putative tumor suppressor, recently identified as ω-amidase. hNit2/ω-amidase plays a crucial metabolic role by catalyzing the hydrolysis of α-ketoglutaramate (the α-keto analog of glutamine) and α-ketosuccinamate (the α-keto analog of asparagine), yielding α-ketoglutarate and oxaloacetate, respectively. Transamination between glutamine and α-keto-γ-methiolbutyrate closes the methionine salvage pathway. Thus, hNit2/ω-amidase links sulfur metabolism to the tricarboxylic acid cycle. To elucidate the catalytic specificity of hNit2/ω-amidase, we performed molecular dynamics simulations on the wild type enzyme and its mutants to investigate enzyme-substrate interactions. Binding free energies were computed to characterize factors contributing to the substrate specificity. The predictions resulting from these computations were verified by kinetic analyses and mutational studies. The activity of hNit2/ω-amidase was determined with α-ketoglutaramate and succinamate as substrates. We constructed three catalytic triad mutants (E43A, K112A, and C153A) and a mutant with a loop 116-128 deletion to validate the role of key residues and the 116-128 loop region in substrate binding and turnover. The molecular dynamics simulations successfully verified the experimental trends in the binding specificity of hNit2/ω-amidase toward various substrates. Our findings have revealed novel structural insights into the binding of substrates to hNit2/ω-amidase. A catalytic triad and the loop residues 116-128 of hNit2 play an essential role in supporting the stability of the enzyme-substrate complex, resulting in the generation of the catalytic products. These observations are predicted to be of benefit in the design of new inhibitors or activators for research involving cancer and hyperammonemic diseases.

Oral ellagic acid attenuated LPS-induced neuroinflammation in rat brain: MEK1 interaction and M2 microglial polarization.

Ellagic acid, the marker component of peels of Punica granatum L., is known traditionally to treat traumatic hemorrhage. In this study, the cellular mechanism underlying ellagic acid-induced anti-inflammation was investigated using lipopolysaccharides (LPSs) as a neuroinflammation inducer. Our in vitro data showed that LPS (1 µg/mL) consistently phosphorylated ERK and induced neuroinflammation, such as elevation in tumor necrosis factor-α (TNF-α) and nitric oxide production in treated BV-2 cells. Incubation of ellagic acid significantly inhibited LPS-induced ERK phosphorylation and subsequent neuroinflammation in treated BV-2 cells. Furthermore, our in vivo study of neuroinflammation employed an intranigral infusion of LPS that resulted in a time-dependent elevation in phosphorylated ERK levels in the infused substantia nigra (SN). Oral administration of ellagic acid (100 mg/kg) significantly attenuated LPS-induced ERK phosphorylation. A four-day treatment of ellagic acid did not alter LPS-induced ED-1 elevation but ameliorated LPS-induced reduction in CD206 and arginase-1 (two biomarkers of M2 microglia). A seven-day treatment of ellagic acid abolished LPS-induced increases in heme-oxygenase-1, cyclo-oxygenase 2, and α-synuclein trimer levels (a pathological hallmark) in the infused SN. At the same time, ellagic acid attenuated LPS-induced increases in active caspase 3 and receptor-interacting protein kinase-3 levels (respective biomarkers of apoptosis and necroptosis) as well as reduction in tyrosine hydroxylase-positive cells in the infused SN. In silico analysis showed that ellagic acid binds to the catalytic site of MEK1. Our data suggest that ellagic acid is capable of inhibiting MEK1-ERK signaling and then attenuated LPS-induced neuroinflammation, protein aggregation, and programmed cell deaths. Moreover, M2 microglial polarization is suggested as a novel antineuroinflammatory mechanism in the ellagic acid-induced neuroprotection.

Homology modeling and monoclonal antibody binding of the Der f 7 dust mite allergen.

The group 7 allergens are important allergenic specificities for mite-sensitive patients and may need to be incorporated into new diagnostic and therapeutic strategies. However, little is known about their biological and structural features. Position-specific iterative BLAST showed that they had strong ancestral homology to two related families of lipid-binding proteins, namely, the bactericidal permeability-increasing (BPI) proteins and the odorant-binding protein. A three-dimensional model of Der f 7 made with the Phyre and SWISS-MODEL homology-modeling servers showed a close match with the human BPI coordinates used for its construction. The binding of the monoclonal antibody HD12 known to block IgE binding could be blocked by the linear sequence (46GILDF50) with a critical role for L48 or F50. These hydrophobic residues were located on a surface loop of the model. The properties of Der f 7 that can be deduced from the model provide avenues for further characterizing these allergens, their IgE binding structures and biological properties that can enhance allergenicity.

Insights into the free energy landscape and salt-controlled mechanism of the conformational conversions between human telomeric G-quadruplex structures.

G-quadruplexes have become attractive drug targets in cancer therapy. However, due to the polymorphism of G-quadruplex structures, it is difficult to experimentally verify the relevant structures of multiple intermediates and transition states in dynamic equilibrium. Hence, understanding the mechanism by which structural conversions of G-quadruplexes occur is still challenging. We conducted targeted molecular dynamics simulation with umbrella sampling to investigate how salt affects the conformational conversion of human telomeric G-quadruplex. Our results explore a unique view into the structures and energy barrier of the intermediates and transition states in the interconversion process. The pathway of G-quadruplex conformational interconversion was mapped out by a free energy landscape, consisting of branched parallel pathways with multiple energy basins. We propose a salt-controlled mechanism that as the salt concentration increases, the conformational conversion mechanism switches from multi-pathway folding to sequential folding pathways. The hybrid-I and hybrid-II structures are intermediates in the basket-propeller transformation. In high-salt solutions, the conformational conversion upon K+ binding is more feasible than upon Na+ binding. The free energy barrier for conformational conversions ranges from 1.6 to 4.6 kcal/mol. Our work will be beneficial in developing anticancer agents.

Hydrogen bonds in membrane proteins.

Hydrogen bonds are essential tie points inside protein structures. They undergo dynamic rupture and rebonding processes on the time scale of tens of picoseconds. Proteins can partially rearrange during such ruptures. In previous work, we performed molecular dynamics simulations of these fluctuating hydrogen bonds. This indicated long-range entropy and energy contributions extending far into the liquid environment. The results showed that the binding of a given hydrogen bond is much reduced as a result of these interactions in water, as is required for biological activity and in very good confirmation of known experimental results. The larger water environment directly interacts with the hydrogen bond essentially due to long-range molecular interactions. Such a substantial lowering of the energy of the hydrogen bond in water brings it into the range of activation by many biological processes ( Sheu et al. Chem. Phys. Lett. 2008 , 462 , 1 - 5 ). Thus, the water medium profoundly increases the rate. Furthermore, very large entropic changes are associated with the rupture of hydrogen bonds in water, whereas no such effects are seen for the isolated molecule. Interestingly, such an increase in rates in water is still accompanied by a large negative change in entropy in the extended solvent environment, and this reduces the rate by some 2 orders of magnitude. Recent molecular dynamics experiments in D(2)O substantiate this model and show a large solvent isotope effect. In this work, we used lipids as the environment for the hydrogen bond and discovered that the energy is also reduced from that found in the isolated molecule, but not as far as in water. On the other hand, we found that no entropy penalty exists for breaking the hydrogen bond in lipids, as seen for water. These two effects compensate, even though the energy is some 2 times larger. The entropic penalty is reduced such that the rate is higher than in water despite the higher energy. This is a significant result for understanding the reactivity and dynamics of proteins in lipids. It should be noted that these are very important solvent effects on entropies and free energies that are not usually reflected in statistical thermodynamic computations for reactants and products. The very long-range effect of the solvent makes substantial contributions to kinetic rate constants and is readily evaluated in this kinetic method. To ignore these long-range environmental effects on the entropy can lead to very spurious results when calculating rates of protein mobilities. Hence, the results not only agree very well with the known hydrogen-bond energies directly as a result of various environmental factors, but even correctly predict a phase transition in the lipid.

Surface Topography Effects of Globular Biomolecules on Hydration Water.

Hydration water serves as a microscopic manifestation of structural stability and functions of biomolecules. To develop bio-nanomaterials in applications, it is important to study how the surface topography and heterogeneity of biomolecules result in their diversity of the hydration dynamics and energetics. We here performed molecular dynamics simulations combined with the steered molecular dynamics and umbrella sampling to investigate the dynamics and escape process associated with the free energy change of water molecules close to a globular biomolecule, i.e., hemoglobin (Hb) and G-quadruplex DNA (GDNA). The residence time, power of long-time tail, and dipole relaxation time were found to display drastic changes within the averaged hydration shell of 3.0-5.0 Å. Compared with bulk water, in the inner hydration shell, the water dipole moment displays a slower relaxation process and is more oriented toward GDNA than toward Hb, forming a hedgehog-like structure when it surrounds GDNA. In particular, a spine water structure is observed in the GDNA narrow groove. The water isotope effect not only prolongs the dynamic time scales of libration motion in the inner hydration shell and the dipole relaxation processes in the bulk but also strengthens the DNA spine water structure. The potential of the mean force profile reflects the integrity of the hydration shell structure and enables us to obtain detailed insights into the structures formed by water, such as the caged H-bond network and the edge bridge structures; it also reveals that local hydration shell free energy (LHSFE) depends on H-bond rupture processes and ranges from 0.2 to 4.2 kcal/mol. Our results demonstrate that the surface topography of a biomolecule influences the integrity of the hydration shell structure and LHSFE. Our studies are able to identify various further applications in the areas of microfluid devices and nano-dewetting on bioinspired surfaces.

Molecular dynamics of hydrogen bonds in protein-D2O: the solvent isotope effect.

We suggest that the H-bond in proteins not only mirrors the motion of hydrogen in its own atomistic setting but also finds its origin in the collective environment of the hydrogen bond in a global lattice of surrounding H2O molecules. This water lattice is being perturbed in its optimal entropic configuration by the motion of the H-bond. Furthermore, bonding interaction with the lattice drop the H-bond energy from some 5 kcal/mol for the pure protein in the absence of H2O, to some 1.6 kcal/mol in the presence of the H2O medium. This low value here is determined in a computer experiment involving MD calculations and is a value close to the generally accepted value for biological systems. In accordance with these computer experiments under ambient conditions, the H-bond energy is seriously depressed, hence confirming the subtle effect of the H2O medium directly interacting with the H-bond and permitting a strong fluxional behavior. Furthermore, water produces a very large change in the entropy of activation due to the hydrogen bond breakage, which affects the rate by as much as 2 orders of magnitude. We also observe that there is an entire ensemble of H-bond structures, rather than a single transition state, all of which contribute to this H-bond. Here the model is tested by changing to D2O as the surrounding medium resulting in a substantial solvent isotope effect. This demonstrates the important influence of the environment on the individual hydrogen bond.

The different modes of binding of the dust mite allergens, Der f 7 and Der p 7, on a monoclonal antibody WH9 contribute to the differential reactivity.

BACKGROUND: Der f 7 and Der p 7 are important house dust mite allergens. An IgE-binding inhibition monoclonal antibody WH9 reacts ten folds stronger against Der p 7 than to Der f 7. The purpose of this study is to identify the antigenic determinant(s) and the structural basis of Der f 7 recognize by WH9. METHODS: WH9-reactive determinant(s) on Der f 7 was identified by immunoblot and immunoblot inhibition. The 3-D binary complex structures of WH9 and the group 7 allergens were simulated with homology modeling and docking methods. RESULTS: WH9 reacted with the Der f 7 f9 fragment. Among the five site-directed Der f 7 mutants, WH9 showed reduced immunoblot reactivity against Der f 7 S156A, D159A and P160A mutants. Only the wild-type protein and the Der f 7 I157A and L158A mutants can inhibit significantly the WH9-binding against Der f 7. The structural model of the Der f 7-WH9 complex suggests residues S156 and D159 of Der f 7 can bind to WH9 via potential hydrogen bonds. CONCLUSION: The structure models of Der f 7-WH9 and Der p 7-WH9 complexes revealed that the differential modes of binding of Der p 7 and Der f 7 allergens on WH9 contribute to the differential reactivity of WH9 against the Der f 7 and the Der p 7 mite allergens.

Distal charge transport in peptides.

Biological systems often transport charges and reactive processes over substantial distances. Traditional models of chemical kinetics generally do not describe such extreme distal processes. In this Review, an atomistic model for a distal transport of information, which was specifically developed for peptides, is considered. Chemical reactivity is taken as the result of distal effects based on two-step bifunctional kinetics involving unique, very rapid motional properties of peptides in the subpicosecond regime. The bifunctional model suggests highly efficient transport of charge and reactivity in an isolated peptide over a substantial distance; conversely, a very low efficiency in a water environment was found. The model suggests ultrafast transport of charge and reactivity over substantial molecular distances in a peptide environment. Many such domains can be active in a protein.

Mechanically Controlled Electron Transfer in a Single-Polypeptide Transistor.

Proteins are of interest in nano-bio electronic devices due to their versatile structures, exquisite functionality and specificity. However, quantum transport measurements produce conflicting results due to technical limitations whereby it is difficult to precisely determine molecular orientation, the nature of the moieties, the presence of the surroundings and the temperature; in such circumstances a better understanding of the protein electron transfer (ET) pathway and the mechanism remains a considerable challenge. Here, we report an approach to mechanically drive polypeptide flip-flop motion to achieve a logic gate with ON and OFF states during protein ET. We have calculated the transmission spectra of the peptide-based molecular junctions and observed the hallmarks of electrical current and conductance. The results indicate that peptide ET follows an NC asymmetric process and depends on the amino acid chirality and α-helical handedness. Electron transmission decreases as the number of water molecules increases, and the ET efficiency and its pathway depend on the type of water-bridged H-bonds. Our results provide a rational mechanism for peptide ET and new perspectives on polypeptides as potential candidates in logic nano devices.

High-temperature ratchets with sawtooth potentials.

The concept of the effective potential is suggested as an efficient instrument to get a uniform analytical description of stochastic high-temperature on-off flashing and rocking ratchets. The analytical representation for the average particle velocity, obtained within this technique, allows description of ratchets with sharp potentials (and potentials with jumps in particular). For sawtooth potentials, the explicit analytical expressions for the average velocity of on-off flashing and rocking ratchets valid for arbitrary frequencies of potential energy fluctuations are derived; the difference in their high-frequency asymptotics is explored for the smooth and cusped profiles, and profiles with jumps. The origin of the difference as well as the appearance of the jump behavior in ratchet characteristics are interpreted in terms of self-similar universal solutions which give the continuous description of the effect. It is shown how the jump behavior in motor characteristics arises from the competition between the characteristic times of the system.

Chemical Inhibition of Human Thymidylate Kinase and Structural Insights into the Phosphate Binding Loop and Ligand-Induced Degradation.

Targeting thymidylate kinase (TMPK) that catalyzes the phosphotransfer reaction for formation of dTDP from dTMP is a new strategy for anticancer treatment. This study is to understand the inhibitory mechanism of a previously identified human TMPK (hTMPK) inhibitor YMU1 (1a) by molecular docking, isothermal titration calorimetry, and photoaffinity labeling. The molecular dynamics simulation suggests that 1a prefers binding at the catalytic site of hTMPK, whereas the hTMPK inhibitors that bear pyridino[d]isothiazolone or benzo[d]isothiazolone core structure in lieu of the dimethylpyridine-fused isothiazolone moiety in 1a can have access to both the ATP-binding and catalytic sites. The binding sites of hTMPK inhibitors were validated by photoaffinity labeling and mass spectrometric studies. Taking together, 1a and its analogues stabilize the conformation of ligand-induced degradation (LID) region of hTMPK and block the catalytic site or ATP-binding site, thus attenuating the ATP binding-induced closed conformation that is required for phosphorylation of dTMP.