Do collective atomic fluctuations account for cooperative effects? Molecular dynamics studies of the U1A-RNA complex

A complete understanding of gene expression relies on a comprehensive understanding of the protein-RNA recognition process. However, the study of protein-RNA recognition is complicated by many factors that contribute to both binding affinity and specificity, including structure, energetics, dynamical motions, and cooperative interactions. Several recent studies have suggested that energetic coupling between residues contributes to formation of the complex between the U1A protein and stem loop 2 of U1 snRNA as a consequence of a cooperative network of interactions. We have performed molecular dynamics simulations on the U1A-RNA complex, including explicit water and counterions, and analyzed the results based on the calculated positional cross-correlations of atomic fluctuations. The results indicate that cross-correlations calculated on a per residue basis agree well with the observed inter-residue cooperativity and predict that the networks identified to date may also be coupled into an extensive hyper-network that reflects the intrinsic rigidity of the RNA recognition motif. In addition, we report a comparison of the MD calculated correlations with the results of a positional covariance analysis based on the sequences of 330 RNA recognition motifs, including U1A. The calculated inter-residue cross-correlations agree very well with the results of the sites exhibiting positional covariance. Collectively, these results strongly support the hypothesis that collective fluctuations contribute to cooperativity and the corresponding observed thermodynamic coupling. Predictions of additional sites in U1A that may be involved in cooperative networks are advanced.     

Solvation effects on alternative nucleophilic substitution reaction paths for chloride/allyl chloride and gamma-methylated congeners

An adiabatic connection method, mPW1PW91(0.581)/6-31G(d), was employed in conjunction with the continuum solvation model SM5.42 to study the effects of solvation on the S(N)1, S(N)2, and syn and anti S(N)2' nucleophilic substitution reactions of chloride anion with allyl chloride and its gamma-methylated analogues Z- and E-crotyl chloride and isoprenyl chloride. The impact of equilibrium solvation on the potential energy surfaces of these systems is large and leads to significant changes in both the geometries and the relative energetics of different reaction pathways for different species. The predicted effects of increased solvent dielectric constant are consistent with available experimental data and provide semiquantitative insights into the relative influence of different solvents on particular properties.     

Application of matrix-assisted laser desorption/ionization to on-line aerosol time-of-flight mass spectrometry

Matrix-assisted laser desorption/ionization (MALDI) mass spectra were obtained from single biological aerosol particles using an aerosol time-of-flight mass spectrometer (ATOFMS). The inlet to the ATOFMS was coupled with an evaporation/condensation flow cell that allowed the aerosol to be coated with matrix material as the sampled stream entered the spectrometer. Mass spectra were generated from aerosol composed either of gramicidin-S or erythromycin, two small biological molecules, or from aerosolised spores of Bacillus subtilis var niger. Three different matrices were used: 3-nitrobenzyl alcohol, picolinic acid and sinapinic acid. A spectrum of gramicidin-S was generated from approximately 250 attomoles of material using a molar ratio of 3-nitrobenzyl alcohol to analyte of approximately 20:1. A single peak, located at 1224 Da, was obtained from the bacterial spores. The washing liquid and extract solution from the spores were analyzed using electrospray mass spectrometry and subsequent MS/MS product ion experiments. This independent analysis suggests that the measured species represents part of the B. subtilis peptidoglycan. The on-line addition of matrix allows quasi-real-time chemical analysis of individual, aerodynamically sized particles, with an overall system residence time of less than 5 seconds. These results suggest that a MALDI-ATOFMS can provide nearly real-time identification of biological aerosols. Copyright 2000 John Wiley & Sons, Ltd.     

Determination of traces of copper by the catalysed peroxodisulphate--bromide--ascorbic acid Landolt reaction

The reaction between peroxodisulphate and bromide has been utilized for the catalytic determination of 0-1 ppm of copper in dissolved samples. The kinetics and mechanism of the uncatalysed and catalysed reaction have been studied, and the most important kinetic parameters determined. Under the experimental conditions described, the determination can be carried out in the presence of large numbers of other ions with an error less than 5%.     

Preparation of nonsymmetrically substituted stilbenes in a one-pot two-step heck strategy using ethene as a reagent

We present here a strategy for the preparation of nonsymmetrically substituted stilbenes using a one-pot two-step double Heck strategy. First a protocol is developed for the selective preparation of a range of styrenes using ethene as the alkene coupling partner. Then conditions are found for the effective coupling of the styrenes with aryl halides using a 1:1 stoichiometric ratio of the two components. The use of the microwave apparatus to perform the reactions offers a convenient method for synthesis as well as for safely, easily, and accurately loading vessels with gaseous reagents.     

Alkoxycarbonylation of aryl iodides using gaseous carbon monoxide and pre-pressurized reaction vessels in conjunction with microwave heating

The microwave-promoted alkoxycarbonylation of aryl iodides using reaction vessels pre-pressurized with carbon monoxide is reported. Reactions are performed using 0.1 mol% palladium acetate as catalyst, DBU as base and are complete within 20-30 min. A range of aryl iodide substrates can be converted to the corresponding esters using this methodology. Primary and secondary alcohols work well whereas a tertiary alcohol substrate proves less reactive. The potential for scale-up of the reaction has also been explored.     

Direct conversion of aryl halides to phenols using high-temperature or near-critical water and microwave heating

The direct conversion of aryl halides to the corresponding phenols has been achieved using microwave heating. High-temperature or near-critical water is used as the solvent in conjunction with a copper catalyst and a mineral base.     

Characterization of the dynamics of an essential helix in the U1A protein by time-resolved fluorescence measurements

The RNA recognition motif (RRM), one of the most common RNA-binding domains, recognizes single-stranded RNA. A C-terminal helix that undergoes conformational changes upon binding is often an important contributor to RNA recognition. The N-terminal RRM of the U1A protein contains a C-terminal helix (helix C) that interacts with the RNA-binding surface of a beta-sheet in the free protein (closed conformation), but is directed away from this beta-sheet in the complex with RNA (open conformation). The dynamics of helix C in the free protein have been proposed to contribute to binding affinity and specificity. We report here a direct investigation of the dynamics of helix C in the free U1A protein on the nanosecond time scale using time-resolved fluorescence anisotropy. The results indicate that helix C is dynamic on a 2-3 ns time scale within a 20 degrees range of motion. Steady-state fluorescence experiments and molecular dynamics simulations suggest that the dynamical motion of helix C occurs within the closed conformation. Mutation of a residue on the beta-sheet that contacts helix C in the closed conformation dramatically destabilizes the complex (Phe56Ala) and alters the steady-state fluorescence, but not the time-resolved fluorescence anisotropy, of a Trp in helix C. Mutation of Asp90 in the hinge region between helix C and the remainder of the protein to Ala or Gly subtly alters the dynamics of the U1A protein and destabilizes the complex. Together these results show that helix C maintains a dynamic closed conformation that is stable to these targeted protein modifications and does not equilibrate with the open conformation on the nanosecond time scale.     

Flash column chromatograms estimated from thin-layer chromatography data

Given a sample mass and TLC data, a spreadsheet has been developed that provides information on the amount of silica gel needed, the optimal fraction size, and the degree of separation to be expected before flash chromatography is attempted. The spreadsheet is the first utility of its kind to accurately estimate the retention volume and band volume of analytes, as well as the fraction numbers expected to contain each analyte, and the resolution between adjacent peaks. This information allows users to select optimal parameters for preparative-scale separations before the flash column itself is attempted; ensuring a successful first separation.