Protein:Ligand binding free energies: A stringent test for computational protein design

A computational protein design method is extended to allow Monte Carlo simulations where two ligands are titrated into a protein binding pocket, yielding binding free energy differences. These provide a stringent test of the physical model, including the energy surface and sidechain rotamer definition. As a test, we consider tyrosyl‐tRNA synthetase (TyrRS), which has been extensively redesigned experimentally. We consider its specificity for its substrate l ‐tyrosine (l ‐Tyr), compared to the analogs d ‐Tyr, p‐acetyl‐, and p‐azido‐phenylalanine (ac‐Phe, az‐Phe). We simulate l ‐ and d ‐Tyr binding to TyrRS and six mutants, and compare the structures and binding free energies to a more rigorous “MD/GBSA” procedure: molecular dynamics with explicit solvent for structures and a Generalized Born + Surface Area model for binding free energies. Next, we consider l ‐Tyr, ac‐ and az‐Phe binding to six other TyrRS variants. The titration results are sensitive to the precise rotamer definition, which involves a short energy minimization for each sidechain pair to help relax bad contacts induced by the discrete rotamer set. However, when designed mutant structures are rescored with a standard GBSA energy model, results agree well with the more rigorous MD/GBSA. As a third test, we redesign three amino acid positions in the substrate coordination sphere, with either l ‐Tyr or d ‐Tyr as the ligand. For two, we obtain good agreement with experiment, recovering the wildtype residue when l ‐Tyr is the ligand and a d ‐Tyr specific mutant when d ‐Tyr is the ligand. For the third, we recover His with either ligand, instead of wildtype Gln. © 2015 Wiley Periodicals, Inc.     

The effects of preservation methods, dyes and acidification on the isotopic values (δ15N and δ13C) of two zooplankton species from the KwaZulu-Natal Bight, South Africa

Stable isotope measurements are an important tool for ecosystem trophic linkage studies. Ideally, fresh samples should be used for isotopic analysis, but in many cases organisms must be preserved and analysed later. In some cases dyes must be used to help distinguish organisms from detritus. Since preservatives and dyes are carbon-based, their addition could influence isotopic readings. This study aims to improve understanding of the effects of sample storage method, dye addition and acidification on the δ(15)N and δ(13)C values of zooplankton (Euphasia frigida and Undinula vulgaris). Zooplankton was collected and preserved by freezing, or by the addition of 5% formalin, 70% ethanol, or 5% formalin with added Phloxine B or Rose Bengal, and stored for 1 month before processing. Samples in 5% formalin and 70% ethanol were also kept and processed after 3 and 9 months to study changes over time. Formalin caused the largest enrichment for δ(13)C and a slight enrichment for δ(15)N, while ethanol produced a slight depletion for δ(13)C, and different effects on δ(15)N depending on the species. In formalin, dyes depleted the δ(13)C values, but had variable effects on δ(15)N, relative to formalin alone. Acidification had no significant effect on δ(15)N or δ(13)C for either species. Long-term storage showed that the effects of the preservatives were species-dependent. Although the effects on δ(15)N varied, a relative enrichment in (13)C of samples occurred with time. This can have important consequences for the understanding of the organic flow within a food web and for trophic studies. .     

Diblock copolymers adsorbed at a water-oil interface

We probe the conformation of a diblock copolymer layer adsorbed at the surface of water-in-oil emulsion droplets at various concentration of a molecular surfactant. The diblock copolymer is made of a hydrophobic polybutadiene part linked to a hydrophilic polyethylene oxide one. The measure provides the equilibrium thickness of the polymer layer that is obtained with two different techniques, i.e. dynamic light scattering and force measurements. The structure of the layer is shown to change from a “mushroom” conformation in which the adsorbed chains form independent Gaussian coils to a conformation where they interact and extend in the continuous phase. The transition from one regime to the other is progressive as the ratio surfactant/polymer bulk concentration varies. Received 5 May 2000 and Received in final form 6 July 2000