Primary Reaction Dynamics of Proteorhodopsin Mutant D97N Observed by Femtosecond Infrared and Visible Spectroscopy†

Femtosecond time‐resolved spectroscopy in the visible and IR range was utilized to study the primary reaction dynamics of the proteorhodopsin (PR) D97N mutant in comparison with wild type PR at different pH values. The analysis of the data obtained in the mid‐IR closely resembles the results for wild type PR. The observation of the first ground state intermediate K is initially obscured by a complex reaction scheme of vibrational relaxation and heating effects, but its spectral signature clearly emerges at long delay times. In the visible range, a biexponential decay of the excited state within 30 ps and the formation of the K photoproduct is observed. The decay time constants derived for the D97N mutant in D₂O are slightly larger than in H₂O due to H/D exchange. This kinetic isotope effect is even less pronounced than for wild type PR at pH 6. These results support the current notion of a pH dependent hydrogen bonding network in the retinal binding pocket of PR and a weaker interaction between the retinal Schiff base and the counter ion complex compared to bacteriorhodopsin.     

Luminescence of ruthenium halide complexes containing a hemilabile phosphine pyrenyl ether ligand

A series of Ru(II) complexes, tcc-RuX2(POC4Pyr-P,O)2 (X = Cl (3), Br (4), I (5)), containing the hemilabile phosphine pyrenyl ether ligand 4-{2-(diphenylphosphino)phenoxy}butylpyrene (POC4Pyr (1)) are reported. The synthesis and spectroscopic properties of both the ligand, POC4pyr (1), and ligand oxide, P(=O)OC4pyr 2, and the solid-state structure of 1 are reported. Complexes 3-5 react rapidly with CO to give complexes ttt-RuX2(CO)2(POC4pyr-P)2 (X = Cl (6), Br (7), I (8)). No pyrene excimer emission is detected from 3-5; however, different intensities of excimer emission are observed for 6-8. The intensity of excimer emission decreases through the series, with 6 showing the most intense response. The emission is solely due to intramolecular pyrene excimers at low concentrations (< or =10(-4) M). Comparison of the UV-vis and steady-state fluorescence spectra shows overlap between the low energy d-d absorption of 7 and 8 with excimer emission (480 nm), suggesting nonradiative energy transfer may be occurring. Once excess CO is removed, complexes 6-8 isomerize to cis-dicarbonyl complexes cct-RuX2(CO)2(POC4Pyr-P)2 (X = Cl (9), Br (10), I (11)). The intensity of excimer emission from 9-11 increases with respect to the excimer emission observed for 6-8, with 9 showing a significant increase in excimer intensity.     

Sequence confirmation of modified oligonucleotides using IRMPD in the external ion reservoir of an electrospray ionization Fourier transform ion cyclotron mass spectrometer

Modified oligonucleotides continue to play an important role as antisense compounds that inhibit the expression of genes associated with metabolic disorders, cancer, and infectious diseases. Because the majority of modifications render these molecules refractory to standard enzymatic sequencing techniques, alternative sequencing methods which are fast and reliable are needed. In this work we explore how sugar and backbone modifications affect fragmentation patterns observed from oligonucleotides which are fragmented by infrared multiple photon dissociation in the external reservoir of an electrospray ionization Fourier transform ion cyclotron mass spectrometer. The modifications influence which fragment types (i.e., a(n)-B versus c(n)) dominate and the ease with which the oligonucleotides are fragmented. General observations for confirming the sequence of oligonucleotides are described.     

A microfluidic device for whole-animal drug screening using electrophysiological measures in the nematode C. elegans

This paper describes the fabrication and use of a microfluidic device for performing whole-animal chemical screens using non-invasive electrophysiological readouts of neuromuscular function in the nematode worm, C. elegans. The device consists of an array of microchannels to which electrodes are attached to form recording modules capable of detecting the electrical activity of the pharynx, a heart-like neuromuscular organ involved in feeding. The array is coupled to a tree-like arrangement of distribution channels that automatically delivers one nematode to each recording module. The same channels are then used to perfuse the recording modules with test solutions while recording the electropharyngeogram (EPG) from each worm with sufficient sensitivity to detect each pharyngeal contraction. The device accurately reported the acute effects of known anthelmintics (anti-nematode drugs) and also correctly distinguished a specific drug-resistant mutant strain of C. elegans from wild type. The approach described here is readily adaptable to parasitic species for the identification of novel anthelmintics. It is also applicable in toxicology and drug discovery programs for human metabolic and degenerative diseases for which C. elegans is used as a model.     

Integration of gas chromatography mass spectrometry methods for differentiating ricin preparation methods

The investigation of crimes involving chemical or biological agents is infrequent, but presents unique analytical challenges. The protein toxin ricin is encountered more frequently than other agents and is found in the seeds of Ricinus communis, commonly known as the castor plant. Typically, the toxin is extracted from castor seeds utilizing a variety of different recipes that result in varying purity of the toxin. Moreover, these various purification steps can also leave or differentially remove a variety of exogenous and endogenous residual components with the toxin that may indicate the type and number of purification steps involved. We have applied three gas chromatography-mass spectrometry (GC-MS) based analytical methods to measure the variation in seed carbohydrates and castor oil ricinoleic acid, as well as the presence of solvents used for purification. These methods were applied to the same samples prepared using four previously identified toxin preparation methods, starting from four varieties of castor seeds. The individual data sets for seed carbohydrate profiles, ricinoleic acid, or acetone amount each provided information capable of differentiating different types of toxin preparations across seed types. However, the integration of the data sets using multivariate factor analysis provided a clear distinction of all samples based on the preparation method, independent of the seed source. In particular, the abundance of mannose, arabinose, fucose, ricinoleic acid, and acetone were shown to be important differentiating factors. These complementary tools provide a more confident determination of the method of toxin preparation than would be possible using a single analytical method.     

Antimicrobial sand via adsorption of cationic Moringa oleifera protein

Moringa oleifera (Moringa) seeds contain a natural cationic protein (MOCP) that can be used as an antimicrobial flocculant for water clarification. Currently, the main barrier to using Moringa seeds for producing potable water is that the seeds release other water-soluble proteins and organic matter, which increase the concentration of dissolved organic matter (DOM) in the water. The presence of this DOM supports the regrowth of pathogens in treated water, preventing its storage and later use. A new strategy has been established for retaining the MOCP protein and its ability to clarify and disinfect water while removing the excess organic matter. The MOCP is first adsorbed and immobilized onto sand granules, followed by a rinsing step wherein the excess organic matter is removed, thereby preventing later growth of bacteria in the purified water. Our hypotheses are that the protein remains adsorbed onto the sand after the functionalization treatment, and that the ability of the antimicrobial functionalized sand (f-sand) to clarify turbidity and kill bacteria, as MOCP does in bulk solution, is maintained. The data support these hypotheses, indicating that the f-sand removes silica microspheres and pathogens from water, renders adhered Escherichia coli bacteria nonviable, and reduces turbidity of a kaolin suspension. The antimicrobial properties of f-sand were assessed using fluorescent (live-dead) staining of bacteria on the surface of the f-sand. The DOM that can contribute to bacterial regrowth was shown to be significantly reduced in solution, by measuring biochemical oxygen demand (BOD). Overall, these results open the possibility that immobilization of the MOCP protein onto sand can provide a simple, locally sustainable process for producing storable drinking water.     

Reactions within fluorobenzene-ammonia heterocluster ions: experiment and theory

Reactions occurring within gas phase fluorobenenze-ammonia heterocluster cations (FC(6)H(5)-(NH(3))(n=1-4)) have been studied through the use of a triple quadrupole mass spectrometer as well as employing density functional theory (DFT). Collision induced dissociation (CID) experiments were conducted in which mass selected cluster ions are accelerated into a cell containing argon gas and the resulting products then subsequently mass analyzed. Two dominate reaction channels are observed. The first is simple evaporative loss of neutral ammonia from the cluster ion. The second involves a substitution reaction occurring within the cluster ion to form the aniline cation, C(6)H(5)NH(2)(+), where the reactivity was found to vary as a function of cluster size. DFT calculations have been performed to both help analyze the structure and the reactivity of these cluster ions. Pronounced differences in activation energies were found that provide an explanation for the observed variation of reactivity as a function of cluster size. An ad hoc model based upon the Arrhenius equation was developed to fit both the experimental collision energy dependence of the reaction and the observed lowering of the reaction barrier to aniline formation as a function of cluster size.     

Substantial improvements in large-scale redocking and screening using the novel HYDE scoring function

The HYDE scoring function consistently describes hydrogen bonding, the hydrophobic effect and desolvation. It relies on HYdration and DEsolvation terms which are calibrated using octanol/water partition coefficients of small molecules. We do not use affinity data for calibration, therefore HYDE is generally applicable to all protein targets. HYDE reflects the Gibbs free energy of binding while only considering the essential interactions of protein-ligand complexes. The greatest benefit of HYDE is that it yields a very intuitive atom-based score, which can be mapped onto the ligand and protein atoms. This allows the direct visualization of the score and consequently facilitates analysis of protein-ligand complexes during the lead optimization process. In this study, we validated our new scoring function by applying it in large-scale docking experiments. We could successfully predict the correct binding mode in 93% of complexes in redocking calculations on the Astex diverse set, while our performance in virtual screening experiments using the DUD dataset showed significant enrichment values with a mean AUC of 0.77 across all protein targets with little or no structural defects. As part of these studies, we also carried out a very detailed analysis of the data that revealed interesting pitfalls, which we highlight here and which should be addressed in future benchmark datasets.     

The mechanostability of isolated focal adhesions is strongly dependent on pH

This report demonstrates that the mechanical stability of focal adhesions exhibits a biphasic and sensitive pH dependence. These studies used isolated focal adhesions, which retain many of the properties of the intracellular structures, including protein composition and force-dependent reinforcement by cytosolic proteins. The focal adhesion structures are least stable to applied force at a pH of 6.4, and significantly more stable at slightly higher and lower pH values. This trend is consistent with previous work that characterized the pH dependence of cell migration and may therefore be relevant to controlling the invasiveness of metastatic cancer cells. This approach is significant because it allows biochemical studies of large protein complexes previously studied only in cell culture, and therefore offers new opportunities for performing mechanistic studies of a range of factors that contribute to focal adhesion stability.     

A performance study of radio-opaque personal protective fabrics for the reduction of transmittance of gamma-rays and neutrons

Commercial radio-opaque combat (CRC) fabrics, for incorporation into personal protective equipment used by first responders and armed forces, are marketed as having the ability to provide a level of protection against specific types of radiation. For a CRC material, a standard combat uniform and a multi-layered chemical, biological, radiological, nuclear (CBRN) protective material, the present work examines chemical composition and radiation protection against gamma-rays and neutron fluxes. Significant reduction in gamma-ray transmittance occurs only for the CRC fabric (46–514 keV) with gamma-ray attenuation coefficients of 3.10 to <0.10 cm² g⁻¹. Reduction in neutron transmittance, for all three fabrics, could not be assessed with certainty as the measured transmittance was obscured by large statistical uncertainties.