N‐methyl‐2‐pyrrolidonium methyl sulfonate acidic ionic liquid as a new dynamic coating for separation of basic proteins by capillary electrophoresis

A simple and economical CE method has been developed for the analysis of four model basic proteins by employing N‐methyl‐2‐pyrrolidonium methyl sulfonate ionic liquid (IL) as the dynamic coating material based on the interaction of both between electrostatic attraction and hydrogen bond, and between the organic cations of IL and the inner surface of bare fused‐silica capillary. The N‐methyl‐2‐pyrrolidonium‐based IL modified capillary not only generated a stable suppressed electroosmotic flow, but also effectively eliminated the wall adsorption of proteins. Several important parameters such as the IL concentration, pH values, and concentrations of the background electrolyte were optimized to improve the separation of basic proteins. Consequently, under the optimum separation conditions, a satisfied separation of basic proteins including lysozyme, cytochrome c, ribonuclease A, and α‐chymotrypsinogen A with theoretical plates ranging from 2.09 × 10⁵ to 4.48 × 10⁵ plates/m had been accomplished within 15 min. The proposed method first illustrated the effect of hydrogen bond between coating material and inner capillary surface on the coating, which should be a new strategy to design and select more effective coating materials to form more stable coatings in CE.     

A microfluidic chip‐based fluorescent biosensor for the sensitive and specific detection of label‐free single‐base mismatch via magnetic beads‐based “sandwich” hybridization strategy

A novel microfluidic chip‐based fluorescent DNA biosensor, which utilized the electrophoretic driving mode and magnetic beads‐based “sandwich” hybridization strategy, was developed for the sensitive and ultra‐specific detection of single‐base mismatch DNA in this study. In comparison with previous biosensors, the proposed DNA biosensor has much more robust resistibility to the complex matrix of real saliva and serum samples, shorter analysis time, and much higher discrimination ability for the detection of single‐base mismatch. These features, as well as its easiness of fabrication, operation convenience, stability, better reusability, and low cost, make it a promising alternative to the SNPs genotyping/detection in clinical diagnosis. By using the biosensor, we have successfully determined oral cancer‐related DNA in saliva and serum samples without sample labeling and any preseparation or dilution with a detection limit of 5.6 × 10^?11 M, a RSD (n = 5) < 5% and a discrimination factor of 3.58–4.54 for one‐base mismatch.     

Direct Blue 71 staining as a destaining‐free alternative loading control method for Western blotting

In Western blotting, a suitable loading control is indispensable for correcting errors in the total amount of loaded protein. Immunodetection of housekeeping proteins and total protein staining have traditionally been used as loading control methods. Direct Blue 71 (DB71) staining—a novel, sensitive, dye‐binding staining method compatible with immunodetection—may offer advantages over these traditional loading control methods. Three common neuroscientific samples (human plasma, human oligodendrocytes, and rat brain) were employed to assess DB71 staining as a loading control method for Western blotting. DB71, CBB, one traditional housekeeping protein, and one protein of interest were comparatively assessed for reliability and repeatability and linear dynamic range over 2.5–40 μg of protein loaded. DB71's effect on the reliability and repeatability and linear dynamic range of immunoreaction were also assessed. Across all three sample types, DB71 was either equivalent or superior to CBB and housekeeping protein‐based methods in terms of reliability and repeatability and linear dynamic range. Across all three sample types, DB71 staining did not impair the reliability and repeatability or linear dynamic range of immunoreaction. Our results demonstrate that the DB71 staining can be used as a destaining‐free alternative loading control method for Western blotting.     

Fluorescent staining of protein in SDS polyacrylamide gels by salicylaldehyde azine

As a noncovalent fluorescence probe, in this study, salicylaldehyde azine (SA) was introduced as a sensitive fluorescence‐based dye for detecting proteins both in 1D and 2D polyacrylamide electrophoresis gels. Down to 0.2 ng of single protein band could be detected within 1 h, which is similar to that of glutaraldehyde‐silver stain, but approximately four times higher than that of SYPRO Ruby fluorescent stain. Furthermore, comparative analysis of the MS compatibility of SA stain with SYPRO Ruby stain indicated that SA stain is compatible with the downstream of protein identification by LC‐MS/MS. Additionally, the probable mechanism of the SA stain was investigated by molecular docking. The results demonstrated that the interaction between SA and protein was mainly contributed by hydrogen bonding and hydrophobic forces.     

High–quality protein backbone reconstruction from alpha carbons using Gaussian mixture models

Coarse‐grained protein structure models offer increased efficiency in structural modeling, but these must be coupled with fast and accurate methods to revert to a full‐atom structure. Here, we present a novel algorithm to reconstruct mainchain models from C traces. This has been parameterized by fitting Gaussian mixture models (GMMs) to short backbone fragments centered on idealized peptide bonds. The method we have developed is statistically significantly more accurate than several competing methods, both in terms of RMSD values and dihedral angle differences. The method produced Ramachandran dihedral angle distributions that are closer to that observed in real proteins and better Phaser molecular replacement log‐likelihood gains. Amino acid residue sidechain reconstruction accuracy using SCWRL4 was found to be statistically significantly correlated to backbone reconstruction accuracy. Finally, the PD2 method was found to produce significantly lower energy full‐atom models using Rosetta which has implications for multiscale protein modeling using coarse‐grained models. A webserver and C++ source code is freely available for noncommercial use from: http://www.sbg.bio.ic.ac.uk/phyre2/PD2_ca2main/ . © 2013 Wiley Periodicals, Inc.     

Computational gibberellin‐binding channel discovery unraveling the unexpected perception mechanism of hormone signal by gibberellin receptor

Gibberellins (GAs) are phytohormones essential for many developmental processes in plants. In this work, fundamental mechanism of hormone perception by receptor GID1 has been studied by performing computational simulations, revealing a new GA‐binding channel of GID1 and a novel hormone perception mechanism involving only one conformational state of GID1. The novel hormone perception mechanism demonstrated here is remarkably different from the previously proposed/speculated mechanism [Murase et al., Nature 2008 , 456, 459] involving two conformational states (“OPEN” and “CLOSED”) of GID1. According to the new perception mechanism, GA acts as a “conformational stabilizer,” rather than the previously speculated “allosteric inducer,” to induce the recognition of protein DELLA by GID1. The novel mechanistic insights obtained in this study provide a new starting point for further studies on the detailed molecular mechanisms of GID1 interacting with DELLA and various hormones and for mechanism‐based rational design of novel, potent growth regulators that target crops and ornamental plants. © 2013 Wiley Periodicals, Inc.     

Rapid parameterization of small molecules using the force field toolkit

The inability to rapidly generate accurate and robust parameters for novel chemical matter continues to severely limit the application of molecular dynamics simulations to many biological systems of interest, especially in fields such as drug discovery. Although the release of generalized versions of common classical force fields, for example, General Amber Force Field and CHARMM General Force Field, have posited guidelines for parameterization of small molecules, many technical challenges remain that have hampered their wide‐scale extension. The Force Field Toolkit (ffTK), described herein, minimizes common barriers to ligand parameterization through algorithm and method development, automation of tedious and error‐prone tasks, and graphical user interface design. Distributed as a VMD plugin, ffTK facilitates the traversal of a clear and organized workflow resulting in a complete set of CHARMM‐compatible parameters. A variety of tools are provided to generate quantum mechanical target data, setup multidimensional optimization routines, and analyze parameter performance. Parameters developed for a small test set of molecules using ffTK were comparable to existing CGenFF parameters in their ability to reproduce experimentally measured values for pure‐solvent properties (<15% error from experiment) and free energy of solvation (±0.5 kcal/mol from experiment). © 2013 Wiley Periodicals, Inc.     

Investigation on the Stability of the Dispersion System of Cross-Linked Polyacrylamide Microspheres

The salt resistance, temperature resistance, and shear stability of a cross-linked polyacrylamide microsphere system are studied by microfiltration, light diffraction analysis, and optical microscopy. The results show that other conditions being equal, the particle diameter of cross-linked polyacrylamide microspheres decreases with increased NaCl concentration. When NaCl concentration is lower than 10,000 mg/L, its effect on the plugging performance of a cross-linked polyacrylamide microsphere system in regard to the nuclear pore membrane is weak in comparison with a linked polymer solution, but the former system has better salt tolerance. Particle diameter decreases with increased swelling temperature. When the swelling temperature is below 90°C, its effect on the plugging performance of the cross-linked polyacrylamide microsphere system in regard to nuclear pore membrane is weak in comparison with the linked polymer solution, but has better temperature tolerance. Particle size shows little change, with shearing rate being increased, while the shape remains about the same and the effect of shearing on the plugging performance of the cross-linked polyacrylamide microsphere dispersion system in regard to the nuclear pore membrane is weak in comparison with the linked polymer solution, but has better shear stability. The salt tolerance, temperature tolerance, and shear stability of microspheres are associated with a particular cross-linked structure.     

The Effects of Alcohol with Different Chain Length on the Phase Equilibria of Nonionic Surfactant System

Addition of alcohol with longer chain length (C₆H₁₃OH, C₈H₁₇OH, and C₁₂H₂₁OH) caused a reduction the cloud point of a commercial nonionic surfactant, Tesgitol (T_15-s-9). The formation of lamellar liquid crystal (LLC) was favored so that isotropic liquid (L₁)-LLC two-phase region became wider with increasing temperature at an appropriate weight ratio of surfactant to alcohol. The isotropic liquid phase/liquid two phase transformation was replaced by a two-phase transformation to isotropic liquid/lamellar liquid crystal at the cloud point for the system without alcohol.     

Determination of the Diffusion Coefficient for SDS Micelle with Different Shape and the Effects of Ethanol by Cyclic Voltammetry Without Probes

Micelle diffusion coefficient of SDS with different micelle shape in aqueous and water-ethanol solutions are determined by cyclic voltammetry without any probe. The diffusion coefficient decreases with as increasing SDS concentration. The first critical micellar concentration is 8.0xl0³, mol 1¹.corresponding to the transformation from premicelle to spherical micelle. The second critical micellar concentration is 5.60x10²and corresponding to transformation from spherical micelle to rod-like micelle. The less the weight ratio of SDS to ethanol is, the larger the diffusion coefficient is. The influence of added water to the micelle solution is almost the same for SDS-ethanol-HaO system with different micelle shape. Mechanism of electrochemical reaction for SDS at platinum electrode is discussed as well.