A practical approach that enables one to calculate the standard free energy of binding from a one-dimensional potential of mean force (PMF) is proposed. Umbrella sampling and the weighted histogram analysis method are used to generate a PMF along the reaction coordinate of binding. At each point, a restraint is applied orthogonal to the reaction coordinate to make possible the determination of the volume sampled by the ligand. The free energy of binding from an arbitrary unbound volume to the restrained bound form is calculated from the ratio of the PMF integrated over the bound region to that of the unbound. Adding the free energy changes from the standard-state volume to the unbound volume and from the restrained to the unrestrained bound state gives the standard free energy of binding. Exploration of the best choice of binding paths is also made. This approach is first demonstrated on a model binding system and then tested on the benzamidine-trypsin system for which reasonable agreement with experiment is found. A comparison is made with other methods to obtain the standard free energy of binding from the PMF.
Pathogen access to host nutrients in infected tissues is fundamental for pathogen growth and virulence, disease progression, and infection control. However, our understanding of this crucial process is still rather limited because of experimental and conceptual challenges. Here, we used proteomics, microbial genetics, competitive infections, and computational approaches to obtain a comprehensive overview of Salmonella nutrition and growth in a mouse typhoid fever model. The data revealed that Salmonella accessed an unexpectedly diverse set of at least 31 different host nutrients in infected tissues but the individual nutrients were available in only scarce amounts. Salmonella adapted to this situation by expressing versatile catabolic pathways to simultaneously exploit multiple host nutrients. A genome-scale computational model of Salmonella in vivo metabolism based on these data was fully consistent with independent large-scale experimental data on Salmonella enzyme quantities, and correctly predicted 92% of 738 reported experimental mutant virulence phenotypes, suggesting that our analysis provided a comprehensive overview of host nutrient supply, Salmonella metabolism, and Salmonella growth during infection. Comparison of metabolic networks of other pathogens suggested that complex host/pathogen nutritional interfaces are a common feature underlying many infectious diseases.
Reactive oxygen and nitrogen species function in host defense via mechanisms that remain controversial. Pathogens might encounter varying levels of these species, but bulk measurements cannot resolve such heterogeneity. We used single-cell approaches to determine the impact of oxidative and nitrosative stresses on individual Salmonella during early infection in mouse spleen. Salmonella encounter and respond to both stresses, but the levels and impact vary widely. Neutrophils and inflammatory monocytes kill Salmonella by generating overwhelming oxidative stress through NADPH oxidase and myeloperoxidase. This controls Salmonella within inflammatory lesions but does not prevent their spread to more permissive resident red pulp macrophages, which generate only sublethal oxidative bursts. Regional host expression of inducible nitric oxide synthase exposes some Salmonella to nitrosative stress, triggering effective local Salmonella detoxification through nitric oxide denitrosylase. Thus, reactive oxygen and nitrogen species influence dramatically different outcomes of disparate Salmonella-host cell encounters, which together determine overall disease progression.
A hybrid quantum mechanical/molecular mechanical
approach is used to elucidate structural and energetic
features of amide hydrolysis by the enzyme papain. The role of the
enzyme in stabilizing the thiolate−imidazolium
ion pair is examined and the potential energy pathway for the
subsequent attack of the cysteine anion and proton
transfer from the imidazolium cation is determined. The reaction
is found to be concerted rather than stepwise, and
the transition state for the reaction is located. The effect of
residue mutations both on the ion pair stability and on
the barrier to amide hydrolysis is explored and found to be in
agreement with experiment. In this work both high-level electronic structure and semiempirical MO methods are used, with
location and characterization of stationary
structures. Rearrangement of the enzyme in response to the
changing electronic structure of the active site is also
considered.
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