Comparison of DNA hydration patterns obtained using two distinct computational methods, molecular dynamics simulation and three-dimensional reference interaction site model theory

Because proteins and DNA interact with each other and with various small molecules in the presence of water molecules, we cannot ignore their hydration when discussing their structural and energetic properties. Although high-resolution crystal structure analyses have given us a view of tightly bound water molecules on their surface, the structural data are still insufficient to capture the detailed configurations of water molecules around the surface of these biomolecules. Thanks to the invention of various computational algorithms, computer simulations can now provide an atomic view of hydration. Here, we describe the apparent patterns of DNA hydration calculated by using two different computational methods: Molecular dynamics (MD) simulation and three-dimensional reference interaction site model (3D-RISM) theory. Both methods are promising for obtaining hydration properties, but until now there have been no thorough comparisons of the calculated three-dimensional distributions of hydrating water. This rigorous comparison showed that MD and 3D-RISM provide essentially similar hydration patterns when there is sufficient sampling time for MD and a sufficient number of conformations to describe molecular flexibility for 3D-RISM. This suggests that these two computational methods can be used to complement one another when evaluating the reliability of the calculated hydration patterns.     

"Drop-and-Sip" fluid handling technique for the reagent-release capillary (RRC)-based capillary-assembled microchip (CAs-CHIP): sample delivery optimization and reagent release behavior in RRC.

The experimental conditions of the sample delivery inside the reagent-release capillary-based capillary-assembled microchip (RRC-based CAs-CHIP) were optimized and the reagent release procedure in the RRC is discussed. Recently, our group introduced the basic concept of the "drop-and-sip" fluid handling technique (Anal. Chem., 2007, 79, 908). A microliter volume of sample solution is dropped on the inlet hole and is sipped into another hole, producing a sample plug flow in the main poly(dimethyl siloxane) (PDMS) channel, concurrently filling each sensing capillary that faces the main PDMS channel. However, the detailed evaluation of the successful sample delivery condition and the reagent release behavior in the RRC has not been fully discussed. Under our experimental conditions, ca. 0.6 - 2.4 s of sample plug-RRC contact time allowed the successful sample introduction into the RRC by capillary force without any reagent leakage or disturbance of the sample plug flow. On the other hand, reagent release behavior inside the RRC is governed by both convective and diffusive mass transport, which leads to a faster mixing time of the sample with reagents immobilized inside the RRC compared to that expected from the simple diffusion alone.     

Simultaneous analysis of ultrafast fluorescence decays of FMN binding protein and its mutated proteins by molecular dynamic simulation and electron transfer theory

Ultrafast fluorescence decays of FMN binding proteins (FBP) from Desulfovibrio vulgaris (Miyazaki F) were analyzed with an electron transfer (ET) theory by Kakitani and Mataga (KM theory). Time-dependent distances among isoalloxazine (Iso) and Trp-32, Tyr-35, and Trp-106 in wild-type FBP (WT), among Iso and Tyr-32, Tyr-35, and Trp-106 in W32Y (Trp-32 was replaced by Tyr-32), and among Iso and Tyr-35 and Trp-106 in W32A (Trp-32 was replaced by Ala-32) were determined by molecular dynamic simulation (MD). Electrostatic energies between Iso anion and all other ionic groups, between Trp-32 cation and all other ionic groups, and between Tyr-32 cation and all other ionic groups were calculated in WT, W32Y, and W32A, from the MD coordinates. ET parameters contained in KM theory, such as frequency (nu 0), a coefficient of the ET process (beta), a critical distance of the ET process ( R 0), standard free energy related to the electron affinity of the excited Iso ( G Iso (0)), and the static dielectric constant in FBP species (epsilon 0), were determined with and without inclusion of the electrostatic energy, so as to fit the calculated fluorescence decays with the observed decays of all FBP species, by a nonlinear least-squares method according to the Marquardt algorithm. In the analyses the parameters, nu 0, beta, and R 0 were determined separately between Trp residues and Tyr residues among all FBP species. Calculated fluorescence intensities with the inclusion of the electrostatic energy fit quite well with the observed ones of all WT, W32Y, and W32A.     

Single-drop analysis of various proteases in a cancer cell lysate using a capillary-assembled microchip

Single-drop analysis of two different real sample solutions (2 μL) while simultaneously monitoring the activity of two sets of ten different proteases on a single microfluidic device is presented. The device, called a capillary-assembled microchip (CAs-CHIP), is fabricated by embedding square glass sensing capillaries (reagent-release capillaries, RRC) in the polydimethylsiloxane (PDMS) lattice microchannel, and used for that purpose. First, the performance reliability was evaluated by measuring the fluorescence response of twenty caspase-3-sensing capillaries on a single CAs-CHIP, and a relative standard deviation of 1.5–8.2 (% RSD, n = 5 or 10) was obtained. This suggests that precise multiplexed protease-activity sensing is possible by using a single CAs-CHIP with multiple RRCs embedded. Then, using a single CAs-CHIP, real sample analysis of the activity of ten different caspases/proteases in cervical cancer (HeLa) cell lysate treated and untreated with the cell-death-inducer drug, doxorubicin, was simultaneously carried out, and a significant difference in enzyme activity between these two samples was observed. These results suggested the usefulness of the CAs-CHIP in the field of drug discovery. Figure Single drop analysis of two real sample solutions including various different proteases using a single microfluidic device was achieved     

New Synthesis Method Using Magnesium Alkoxides as Carrier Materials for Ziegler‐Natta Catalysts with Spherical Morphology

In this study, novel carrier materials were synthesized by addition of metal dihalide compounds in the synthesis reaction of magnesium diethoxide using metallic magnesium, ethanol and iodine. Poly(propylene) polymerizations were then investigated with the MgCl₂‐supported TiCl₄ catalysts using these carrier materials. As results, magnesium diethoxide with extremely large particle sizes and spherical shapes were obtained and the angles of repose of PP particles obtained by using their catalysts as a flowability index showed high values. Furthermore, in order to confirm key points for excellent catalyst performance from detailed characterizations, SEM images, compositions and WAXS were measured.

     

Synthesis and Helical Structure of Poly(1‐methylpropargyl ester)s with Various Side Chains

Optically active 1‐methylpropargyl esters bearing various substituents were polymerized with [(nbd)Rh]⁺[η⁶‐C₆H₅B(C₆H₅)₃]^? (nbd=norbornadiene) as a catalyst to afford the corresponding poly(1‐methylpropargyl ester)s with moderate molecular weights in good yields. The polymers have a cis‐stereoregular structure, which was determined by ¹H NMR spectroscopy. Large optical rotations and clear CD signals demonstrated that all these polymers take on a helical structure with a predominantly one‐handed screw sense. The polymers exhibited large viscosity indices in the range 1.14–1.75. Chiral amplification was observed in R/S copolymerization. Conformational analysis revealed that the polymers form a tightly twisted helical structure with a dihedral angle of 70° at the single bond of the main chain.     

Semi-synthesis of an artificial scandium(iii) enzyme with a β-helical bio-nanotube

We have succeeded in preparing semi-synthesized proteins bound to Sc(3+) ion which can promote an epoxide ring-opening reaction. The Sc(3+) binding site was created on the surface of [(gp5βf)(3)](2) (N. Yokoi et al., Small, 2010, 6, 1873) by introducing a cysteine residue for conjugation of a bpy moiety using a thiol-maleimide coupling reaction. Three cysteine mutants [(gp5βf_X)(3)](2) (X = G18C, L47C, N51C) were prepared to introduce a bpy in different positions because it had been reported that Sc(3+) ion can serve as a Lewis-acid catalyst for an epoxide ring-opening reaction upon binding of epoxide to bpy and two -ROH groups. G18C_bpy with Sc(3+) can accelerate the rate of catalysis of the epoxide ring-opening reaction and has the highest rate of conversion among the three mutants. The value is more than 20 times higher than that of the mixtures of [(gp5βf)(3)](2)/2,2'-bipyridine and l-threonine/2,2'-bipyridine. The elevated activity was obtained by the cooperative effect of stabilizing the Sc(3+) coordination and accumulation of substrates on the protein surface. Thus, we expect that the semi-synthetic approach can provide insights into new rational design of artificial metalloenzymes.     

Total Synthesis of (+)-Sch 725680: Inhibitor of Mammalian A-, B-, and Y-Family DNA Polymerases

The total synthesis of (+)-Sch 725680, a member of the hydrogenated azaphilone family, has been accomplished. The synthesis confirmed the absolute configuration and biological activities of the natural product. A key reaction to construct a hydrogenated azaphilone core skeleton is a Ti-mediated aldol reaction.     

Water adsorption-desorption isotherms of two-dimensional hexagonal mesoporous silica around freezing point

Zr-doped mesoporous silica with a diameter of approximately 3.8 nm was synthesized via an evaporation-induced self-assembly process, and the adsorption-desorption isotherms of water vapor were measured in the temperature range of 263-298 K. The measured adsorption-desorption isotherms below 273 K indicated that water confined in the mesopores did not freeze at any relative pressure. All isotherms had a steep curve, resulting from capillary condensation/evaporation, and a pronounced hysteresis. The hysteresis loop, which is associated with a delayed adsorption process, increased with a decrease in temperature. Furthermore, the curvature radius where capillary evaporation/condensation occurs was evaluated by the combined Kelvin and Gibbs-Tolman-Koening-Buff (GTKB) equations for the modification of the interfacial tension due to the interfacial curvature. The thickness of the water adsorption layer for capillary condensation was slightly larger, whereas that for capillary evaporation was slightly smaller than 0.7 nm.