Tracking Protein S‐Fatty Acylation with Proteomics

Recently, Hang and co‐workers developed “acyl‐PEG exchange” (APE), which allows the investigation of protein S‐fatty acylation with mass‐tag labelling and gel electrophoresis, methods that are accessible to any biochemistry laboratory.     

Structure and Substrate Recognition of the Bottromycin Maturation Enzyme BotP

The bottromycins are a family of highly modified peptide natural products, which display potent antimicrobial activity against Gram‐positive bacteria, including methicillin‐resistant Staphylococcus aureus. Bottromycins have recently been shown to be ribosomally synthesized and post‐translationally modified peptides (RiPPs). Unique amongst RiPPs, the precursor peptide BotA contains a C‐terminal “follower” sequence, rather than the canonical N‐terminal “leader” sequence. We report herein the structural and biochemical characterization of BotP, a leucyl‐aminopeptidase‐like enzyme from the bottromycin pathway. We demonstrate that BotP is responsible for the removal of the N‐terminal methionine from the precursor peptide. Determining the crystal structures of both apo BotP and BotP in complex with Mn²⁺ allowed us to model a BotP/substrate complex and to rationalize substrate recognition. Our data represent the first step towards targeted compound modification to unlock the full antibiotic potential of bottro‐ mycin.     

Structure–Activity Relationships of 2‐Sufonylpyrimidines as Quorum‐Sensing Inhibitors to Tackle Biofilm Formation and eDNA Release of Pseudomonas aeruginosa

Drug‐resistant Pseudomonas aeruginosa (PA) strains are on the rise, making treatment with current antibiotics ineffective. Hence, circumventing resistance or restoring the activity of antibiotics by novel approaches is of high demand. Targeting the Pseudomonas quinolone signal quorum sensing (PQS‐QS) system is an intriguing strategy to abolish PA pathogenicity without affecting the viability of the pathogen. Herein we report the structure–activity relationships of 2‐sulfonylpyrimidines, which were previously identified as dual‐target inhibitors of the PQS receptor PqsR and the PQS synthase PqsD. The SAR elucidation was guided by a combined approach using ligand efficiency and ligand lipophilicity efficiency to select the most promising compounds. In addition, the most effective inhibitors were rationally modified by the guidance of QSAR using Hansch analyses. Finally, these inhibitors showed the capacity to decrease biofilm mass and extracellular DNA, which are important determinants for antibiotic resistance.     

Applications of Two-Dimensional 1H NMR Methods to Photo-Chemically Induced Dynamic Nuclear Polarisation Spectroscopy

Photo-CIDNP is a useful technique to identify surface residues in proteins and to assign resonances in their ¹H NMR spectra by the spectral simplification that is obtained. In spite of this simplification, conventional photo-CIDNP spectra can be quite complex due to spectral overlap. For this reason two-dimensional (2D) methods are welcome. We present combinations of photo-CIDNP with 2D J-correlated spectroscopy (CIDNP-COSY) and with 2D NOE spectroscopy (CIDNP-NOESY). In these experiments we insert a saturation pulse sequence and a short laser-irradiation period at the beginning of the preparation period to generate CIDNP, leaving the remaining of the pulse scheme essentially unaltered. The CIDNP-COSY and CIDNP-NOESY spectra of a hen egg-white (HEW) lysozyme are presented. The CIDNP-NOESY spectrum of lysozyme is compared with the results from 1D cross-polarisation experiments on the same protein. CIDNP-NOESY spectra of lysozyme with and without inhibitor NAG are compared.     

Adsorption on activated carbon in countercurrent flow: An experimental study

The work describes adsorption experiments from aqueous solutions on activated carbon using a counter-current adsorber. Phenol and paranitrophenol were used for single component adsorption and molasses colour for multicomponent adsorption experiments. Isotherms were determined using simple equilibrium experiments. Three different techniques were used for determination of the kinetics. These were finite bath, infinite bath and fixed bed experiments. A mathematical model for countercurrent adsorption was tested. The model includes the effect of film diffusion and pore diffusion as well as surface diffusion, and can handle systems with a nonlinear isotherms. Kinetic data in the form of an average diffusivity in the carbon determined from the finite bath experiments and isotherm data did not predict the operation of the countercurrent column well. Data from the fixed bed experiments predicted countercurrent column operation much better. This is deemed to be due to inhomogeneities in the carbon used, resulting in a fairly quick initial uptake of the main part of the solute and a slower uptake of a minor amount.     

Significance of low water—solid ratio and temperature on the physico-mechanical characteristics of hydrates of tricalcium aluminate

Hydration characteristics are reported of tricalcium aluminate studied at 20 and 80 °C using water-solid ratios of 0.2 and 1.0. Hydration products were subjected to differential thermal analysis, thermogravimetric analysis, scanning electron microscopy, length change, porosity and pore size distribution, and micro-hardness measurements. Hydration proceeds faster at 80 °C than at 20 °C and the cubic C₃AH₆ phase is detected in the first few seconds. At 20 °C hydration occurs with the initial formation of the hexagonal phases and expansion is continuous. At higher temperatures and a water-solid ratio of 0.2, after an initial small expansion, the dimensional change is low. The product at 80 °C develops more than fourfold the hardness value developed at 20 °C. After 2 days of hydration, porosity, per cent C₃AH₆ and microhardness are 15.2%, 83% and 38.9 kg/mm²; corresponding values for the product obtained at 20 °C are 21.5%, 75% and 9.1 kg/mm². Microstructural examination of the material formed at 80 °C indicates a closely welded and continuous network of the cubic phase. These results reveal that at a low water-solid ratio and higher temperatures the formation of the cubic phase from C₃A results in an enhancement of strength.