Oxygen as a paramagnetic probe of clustering and solvent exposure in folded and unfolded states of an SH3 domain

The N-terminal SH3 domain of the Drosophila modular protein Drk undergoes slow exchange between a folded (Fexch) and highly populated unfolded (Uexch) state under nondenaturing buffer conditions, enabling both Fexch and Uexch states to be simultaneously monitored. The addition of dissolved oxygen, equilibrated to a partial pressure of either 30 atm or 60 atm, provides the means to study solvent exposure with atomic resolution via 13C NMR paramagnetic shifts in 1H,13C HSQC (heteronuclear single quantum coherence) spectra. Absolute differences in these paramagnetic shifts between the Fexch and Uexch states allow the discrimination of regions of the protein which undergo change in solvent exposure upon unfolding. Contact with dissolved oxygen for both the Fexch and Uexch states could also be assessed through 13C paramagnetic shifts which were normalized based on the corresponding paramagnetic shifts seen in the free amino acids. In the Fexch state, the 13C nuclei belonging to the hydrophobic core of the protein exhibited very weak normalized paramagnetic shifts while those with greater solvent accessible surface area exhibited significantly larger normalized shifts. The Uexch state displayed less varied 13C paramagnetic shifts although distinct regions of protection from solvent exposure could be identified by a lack of such shifts. These regions, which included Phe9, Thr12, Ala13, Lys21, Thr22, Ile24, Ile27, and Arg38, overlapped with those found to have residual nativelike and non-native structures in previous studies and in some cases provided novel information. Thus, the paramagnetic shifts from dissolved oxygen are highly useful in the study of a transient structure or clustering in disordered systems, where conventional NMR measurements (couplings, chemical shift deviations from random coil values, and NOEs) may give little information.     

A photoimmobilisation strategy that maximises exploration of chemical space in small molecule affinity selection and target discovery

We show that the use of multiple photochemistries is necessary to ensure diverse immobilisation of small molecules for binding of polypeptides using phage display and antibody libraries.     

Tight-binding molecular dynamics study of the role of defects on carbon nanotube moduli and failure

We performed tight-binding molecular dynamics on single-walled carbon nanotubes with and without a variety of defects to study their effect on the nanotube modulus and failure through bond rupture. For a pristine (5,5) nanotube, Young's modulus was calculated to be approximately 1.1 TPa, and brittle rupture occurred at a strain of 17% under quasistatic loading. The predicted modulus is consistent with values from experimentally derived thermal vibration and pull test measurements. The defects studied consist of moving or removing one or two carbon atoms, and correspond to a 1.4% defect density. The occurrence of a Stone-Wales defect does not significantly affect Young's modulus, but failure occurs at 15% strain. The occurrence of a pair of separated vacancy defects lowers Young's modulus by approximately 160 GPa and the critical or rupture strain to 13%. These defects apparently act independently, since one of these defects alone was independently determined to lower Young's modulus by approximately 90 GPa, also with a critical strain of 13%. When the pair of vacancy defects adjacent, however, Young's modulus is lowered by only approximately 100 GPa, but with a lower critical strain of 11%. In all cases, there is noticeable strain softening, for instance, leading to an approximately 250 GPa drop in the apparent secant modulus at 10% strain. When a chiral (10,5) nanotube with a vacancy defect was subjected to tensile strain, failure occurred through a continuous spiral-tearing mechanism that maintained a high level of stress (2.5 GPa) even as the nanotube unraveled. Since the statistical likelihood of defects occurring near each other increases with nanotube length, these studies may have important implications for interpreting the experimental distribution of moduli and critical strains.     

Multi-gigabit WDM optical networking for next generation avionics system communications

It is envisaged that photonic networking will play a significant role in improving performance and reliability in both civil and military avionics systems. Of all the available photonic multiplexing technologies, wavelength-division multiplexing (WDM) has been the primary focus of attention within mainstream telecommunications offering increased throughput at a reasonable cost, with scope for enhanced routing flexibility, connectivity and network survivability. A direct mapping of techniques and devices from the maturing telecommunications sector is, however, not possible because of the stringent requirements of systems operating in the hostile aerospace environment. This paper gives an outline of these requirements and discusses, in detail, the design and development of a multi-gigabit, broadband optical WDM network architecture, specifically for use on aerospace platforms. The paper will also discuss a key element in the system, the arrayed-waveguide grating (AWG) wavelength multiplexing component, which has been designed to allow operation over the full military temperature specification without environmental conditioning.     

Chitosan loaded with silver nanoparticles,CS‐AgNPs,using thymus syriacus,wild mint,and rosemary essential oil extracts as reducing and capping agents

The present study describes the green method for the preparation of chitosan loaded with silver nanoparticles (CS‐AgNPs) in the presence of 3 different extracted essential oils. The essential oils play dual roles as reductant and capping agents. The reducing power and DPPH (2,2‐diphenyl‐1‐picrylhydrazyl) assay for the 3 essential oils—Thymus syriacus (T), wild mint (M), and rosemary (R)—have been reported. The preparation of CS‐AgNPs was performed by 2 steps. The 3 previously extracted essential oils have been used as reducing and capping agent in the first step, while in the second step, silver nanoparticles were integrated in chitosan. The integration of AgNPs in the structure of chitosan was confirmed by ultraviolet‐visible, Fourier transform infrared spectroscopy, scanning electron microscopy techniques, and energy dispersive X‐ray. Surface plasmon resonance confirmed the formation of CS‐AgNPs with maximum absorbance at λ_max between 405 ‐ 410 and 410 ‐ 430 nm for colloidal and films of CS‐AgNPs, respectively. The intensity of bands at 3408 cm^?1 in the fourier transform infrared spectroscopy measurements was decreased substantially and shifted slightly to lower frequency (?υ = 43 cm^?1). Scanning electron microscopy shows a spherical morphology of AgNPs with size of 62 nm for both colloidal and film samples, and energy dispersive X‐ray analysis shows peaks confirming AgNPs formation.     

Enantioselective determination of modafinil in pharmaceutical formulations by capillary electrophoresis, and computational calculation of their inclusion complexes

A fast capillary electrophoretic method is described for the separation and determination of the enantiomers of the novel wake-promoting agent, modafinil. Several parameters affecting the separation were studied, including the type and concentration of chiral selector, buffer pH, buffer concentration, voltage and temperature. Good chiral separation of the racemic mixture was achieved in less than 5 min with resolution factor Rs?=?2.51, using a bare fused-silica capillary and a background electrolyte (BGE) of 25 mM H₃PO₄?1 M tris solution; pH 8.0; containing 30 mg mL^?1 of sulfated-β-cyclodextrin (S-β-CD). The separation was carried out in normal polarity mode at 25 ^?C, 18 kV and using hydrostatic injection. Acceptable validation criteria for selectivity, linearity, precision, and accuracy were included. The developed method was successfully applied to the assay of enantiomers of modafinil in pharmaceutical formulations. The computational calculations for the enantiomeric inclusion complexes rationalized the reasons for the different migration times between the modafinil enantiomers.     

Characterization of a single molecule DNA switch in free solution

We have studied a donor-acceptor fluorophore-labeled DNA switch where the acceptor is Alexa-647, a carbocyanine dye, in solution at the single molecule level to elucidate the fluorescence switching mechanism. The acceptor, which is in an initial high fluorescence trans state, undergoes a photoisomerization reaction resulting in two additional states during its sub-millisecond transit across the probe volume. These two states are assigned to a nonfluorescent triplet trans state that strongly quenches the donor emission and a singlet cis state that blocks the fluorescence resonance energy transfer (FRET) pathway and gives rise to donor-only fluorescence. The formation of these states is faster than the transit time, so that all three states are approximately equally populated under our experimental conditions. The acceptor dye can stick to the DNA in all these states, with the rate of unsticking determining the rate of isomerization into the other states. Measurement of the rate of change of the FRET signal therefore provides information about the fluorophore-DNA intramolecular dynamics. These results explain the large zero peak in the proximity ratio, often seen in single molecule FRET experiments, and suggest that photoinduced effects may be important in single molecule FRET experiments using carbocyanine dyes. They also suggest that for fast photoinduced switching the interactions of the acceptor dye with the DNA and other surfaces should be prevented.     

Multifrequency simulations of the EPR spectra of lipid spin labels in membranes

Simulations are performed of 34- and 9-GHz EPR spectra, together with 94-GHz EPR spectra, from phospholipid probes spin-labelled at the C4-C14 positions of the sn-2 chain, in liquid-ordered and gel-phase membranes of dimyristoyl phosphatidylcholine with high and low cholesterol contents. The multifrequency simulation strategy involves: (i) obtaining partially averaged spin-Hamiltonian tensors from fast-motional simulations of the 94-GHz spectra; (ii) performing slow-motional simulations of the 34- and 9-GHz spectra by using these pre-averaged tensors with the stochastic Liouville formalism; (iii) constructing, by simulation, slow-motional calibrations for the differences, DeltaA(zz)(qx) and Deltag(zz)(qx), in effective A(zz)-hyperfine splittings and g(zz)-values between 34- (or 94-GHz) and 9-GHz spectra; (iv) using such calibrations for DeltaA(zz)(qx) and Deltag(zz)(qx) and dynamic parameters from stage (ii) as a guide to adjust the extent of pre-averaging of the spin-Hamiltonian tensors; and (v) repeating the 34- and 9-GHz simulations of stage (ii). By using this scheme it is possible to obtain consistent values of the rotational diffusion coefficients, D(R perpendicular) and D(R//), and the long-axis order parameter, S(zz), that characterize the slow axial motion of the lipid chains, from spectra at both 34 and 9GHz. Inclusion of spectra at 34GHz greatly improves precision in determining the D(R//) element of the slow diffusion tensor in these systems.     

First results from the CRIS experiment

The ability to study rare isotopes with techniques such as mass spectrometry and laser spectroscopy is often prevented by low production rates and large isobaric contamination. This has necessitated the development of novel beam cleaning techniques that can efficiently isolate the isotope of interest. The Collinear Resonance Ionization Spectroscopy (CRIS) experiment at ISOLDE, achieves this by resonantly ionizing a bunched atom beam in a region of ultra high vacuum. This method is motivated by the need to measure the hyperfine structure and isotope shift at the extremes of isospin where typical production rates drop to 1 atom/s. The technique also offers the ability to purify an ion beam and even select long-lived isomeric states (> 1 ms) from the ground state, which can be subsequently studied by decay spectroscopy or mass spectrometry experiments. This paper will report on the successful commissioning of the CRIS beam line and the recent laser spectroscopy results and laser assisted nuclear decay spectroscopy on the neutron deficient francium isotopes.