A combined far-infrared spectroscopic and electrochemical approach for the study of iron-sulfur proteins

Herein, we present the development of a far-infrared spectroscopic approach for studying metalloenzyme active sites in a redox-dependent manner. An electrochemical cell with 5 mm path and based on silicon windows was found to be appropriate for the measurement of aqueous solutions down to 200 cm(-1) . The cell was probed with the infrared redox signature of the metal-ligand vibrations of different iron-sulfur proteins. Each Fe-S cluster type was found to show a specific spectral signature. As a common feature, a downshift of the frequency of the Fe-S vibrations was seen upon reduction, in line with the increase of the Fe-S bond. This downshift was found to be fully reversible. Electrochemically induced FTIR difference spectroscopy in the far infrared is now possible, opening new perspectives on the understanding of metalloproteins in function of the redox state.     

Ultra rapid prototyping of microfluidic systems using liquid phase photopolymerization

We present a method for the ultra rapid prototyping of microfluidic systems using liquid phase photopolymerization, requiring less than 5 min from design to prototype. Microfluidic device fabrication is demonstrated in a universal plastic or glass cartridge. The method consists of the following steps: introduction of liquid prepolymer into the cartridge, UV exposure through a mask to define the channel geometry, removal of unpolymerized prepolymer, and a final rinse. Rapidly fabricated masters for polydimethylsiloxane micromolding are also demonstrated. The master making process is compared to SU-8 50 photoresist processes. Press-on connectors are developed and demonstrated. All materials used are commercially available and low cost. An extension of these methods (mix and match) is presented that allows for maximal design flexibility and integration with a variety of existing fluidic geometries, components, and processes.     

Resonant inelastic x-ray scattering of methyl chloride at the chlorine K edge

We present a combined experimental and theoretical study of isolated CH(3)Cl molecules using resonant inelastic x-ray scattering (RIXS). The high-resolution spectra allow extraction of information about nuclear dynamics in the core-excited molecule. Polarization-resolved RIXS spectra exhibit linear dichroism in the spin-orbit intensities, a result interpreted as due to chemical environment and singlet-triplet exchange in the molecular core levels. From analysis of the polarization-resolved data, Cl 2p(x, y) and 2p(z) electronic populations can be determined.     

Determining a 'safe' high-mass limit in matrix-assisted laser desorption/ionisation time-of-flight mass spectra of coal derived materials with reference to instrument noise

Three methods for determining a 'safe' estimate for high-mass limits of MALDI spectra of coal derived liquids were explored, using a sample of coal-tar pitch and its pyridine-insoluble fraction. Co-addition of increasing numbers of single-shot spectra (10, 30, 50 and 100 pulses) showed visually observable reductions in noise levels, consistent with robust and statistically meaningful signals. Three separate types of post-acquisition calculation were used to identify high-mass limits of the spectra. (i) A literature method indicated high-mass limits similar to those observed visually-as a shift from baseline at the highest masses, nearly 350 000 u for the coal tar pitch and about 390 000 u for its pyridine insoluble fraction. (ii) Comparing instrument signal with pre-selected multiples of the standard deviation, upper mass estimates of between 40-60 000 u for the coal-tar pitch and about 95 000 u for its pyridine-insoluble fraction were found. (iii) Calculation of the slope was used to identify 'lift-off' of the spectrum from baseline. The angle between the smoothed spectrum and the baseline was matched to a pre-selected value (e.g. 0.5 degrees and 1 degrees ). However, the arbitrary specification of the key parameter did not establish this last method on a firm basis. The choice of a criterion for estimating high-mass limits of MALDI spectra remains a semi-quantitative procedure; a reasonably conservative high-mass limit may be estimated by comparison of signal with five times the standard deviation. However, evaluation of size exclusion chromatograms of the present samples using polystyrene standards suggests that molecular mass distributions of pitch samples arrived at by MALDI mass spectrometry are, at least partly, determined by the limitations of available instruments. Copyright 1999 John Wiley & Sons, Ltd.     

D3R Grand Challenge 4: ligand similarity and MM-GBSA-based pose prediction and affinity ranking for BACE-1 inhibitors

The Drug Design Data Resource (D3R) Grand Challenges present an opportunity to assess, in the context of a blind predictive challenge, the accuracy and the limits of tools and methodologies designed to help guide pharmaceutical drug discovery projects. Here, we report the results of our participation in the D3R Grand Challenge 4 (GC4), which focused on predicting the binding poses and affinity ranking for compounds targeting the $$\beta$$-amyloid precursor protein (BACE-1). Our ligand similarity-based protocol using HYBRID (OpenEye Scientific Software) successfully identified poses close to the native binding mode for most of the ligands with less than 2 Å RMSD accuracy. Furthermore, we compared the performance of our HYBRID-based approach to that of AutoDock Vina and DOCK 6 and found that using a reference ligand to guide the docking process is a better strategy for pose prediction and helped HYBRID to perform better here. We also conducted end-point free energy estimates on molecules dynamics based ensembles of protein-ligand complexes using molecular mechanics combined with generalized Born surface area method (MM-GBSA). We found that the binding affinity ranking based on MM-GBSA scores have poor correlation with the experimental values. Finally, the main lessons from our participation in D3R GC4 are: (i) the generation of the macrocyclic conformers is a key step for successful pose prediction, (ii) the protonation states of the BACE-1 binding site should be treated carefully, (iii) the MM-GBSA method could not discriminate well between different predicted binding poses, and (iv) the MM-GBSA method does not perform well at predicting protein–ligand binding affinities here.