muramidase and acetamide

We apply a free energy perturbation simulation method, free energy perturbation/replica exchange with solute tempering, to two modifications of protein-ligand complexes that lead to significant conformational changes, the first in the protein and the second in the ligand. The approach is shown to facilitate sampling in these challenging cases where high free energy barriers separate the initial and final conformations and leads to superior convergence of the free energy as demonstrated both by consistency of the results (independence from the starting conformation) and agreement with experimental binding affinity data. The second case, consisting of two neutral thrombin ligands that are taken from a recent medicinal chemistry program for this interesting pharmaceutical target, is of particular significance in that it demonstrates that good results can be obtained for large, complex ligands, as opposed to relatively simple model systems. To achieve quantitative agreement with experiment in the thrombin case, a next generation force field, Optimized Potentials for Liquid Simulations 2.0, is required, which provides superior charges and torsional parameters as compared to earlier alternatives.

Topics: Acetamides; Bacteriophage T4; Benzene; Binding Sites; Computer Simulation; Crystallography, X-Ray; Ligands; Models, Molecular; Muramidase; Mutant Proteins; Protein Binding; Protein Conformation; Proteins; Thermodynamics; Thrombin; Valine; Xylenes

The mechanism of irreversible inactivation of lysozyme at neutral pH at 100 degrees C, and effects of additives on the inactivation were investigated. The thermoinactivation of lysozyme at neutral pH was caused by intra- and intermolecular disulfide exchange and the production of irreversibly denatured lysozyme, which was destabilized by multiple chemical reactions other than disulfide exchange. In addition, independently, deamidation slightly affected the inactivation by causing a decrease of electrostatic interaction between positive charges of lysozyme and negative charges of the bacterial cell wall. As for the effects of additives on the inactivation, a small amount of copper ion suppressed intra- and intermolecular disulfide exchange by catalyzing air oxidation of heat-induced trace amounts of free thiols, and organic reagents (acetamide, ethanol, and glycerol) changed the mechanism of the inactivation to that under acidic conditions by shifting the pKa values of dissociable residues and also suppressed intermolecular disulfide exchange by decreasing hydrophobic interactions.

Topics: Acetamides; Animals; Chickens; Copper; Disulfides; Ethanol; Female; Glycerol; Hot Temperature; Hydrogen-Ion Concentration; Kinetics; Micrococcus luteus; Muramidase; Sulfhydryl Compounds; Thermodynamics