Cycloaddition of Phenyltriazolinedione with Carbazole-Alkynes and Yne-Carbamates to Access Diazacyclobutenes.

Previously, we described the synthesis of stable, bicyclic examples of the rather rare diazacyclobutene (DCB) motif by means of a cycloaddition between triazolinediones and electron-rich thiolated alkynes. Here, we report the investigation of the cycloaddition of triazolinediones with related electron-rich yne-carbamates and carbazole-alkynes. Bicyclic DCBs arising from yne-carbamates were isolated in 8-65% yield, while those arising from carbazole-alkynes were isolated in 28-59% yield.

Enolase inhibitors as therapeutic leads for infection.

Infections with the pathogenic free-living amoebae can lead to life-threatening illnesses including catastrophic primary amebic meningoencephalitis (PAM). Efficacious treatment options for these infections are lacking and the mortality rate remains >95% in the US. Glycolysis is very important for the infectious trophozoite lifecycle stage and inhibitors of glucose metabolism have been found to be toxic to the pathogen.

Glucose metabolism in the pathogenic free-living amoebae: Tempting targets for treatment development.

Pathogenic free-living amoebae (pFLA) are single-celled eukaryotes responsible for causing intractable infections with high morbidity and mortality in humans and animals. Current therapeutic approaches include cocktails of antibiotic, antifungal, and antimicrobial compounds. Unfortunately, the efficacy of these can be limited, driving the need for the discovery of new treatments.

A cathepsin C-like protease mediates the post-translation modification of secretory proteins for optimal invasion and egress.

Microbial pathogens use proteases for their infections, such as digestion of proteins for nutrients and activation of their virulence factors. As an obligate intracellular parasite, must invade host cells to establish its intracellular propagation. To facilitate invasion, the parasites secrete invasion effectors from microneme and rhoptry, two unique organelles in apicomplexans.

Exploring the interactive mechanism of acarbose with the amylase SusG in the starch utilization system of the human gut symbiont Bacteroides thetaiotaomicron through molecular modeling.

The α-amylase, SusG, is a principal component of the Bacteroides thetaiotaomicron (Bt) starch utilization system (Sus) used to metabolize complex starch molecules in the human gastrointestinal (GI) tract. We previously reported the non-microbicidal growth inhibition of Bt by the acarbose-mediated arrest of the Sus as a potential therapeutic strategy. Herein, we report a computational approach using density functional theory (DFT), molecular docking, and molecular dynamics (MD) simulation to explore the interactive mechanism between acarbose and SusG at the atomic level in an effort to understand how acarbose shuts down the Bt Sus.

A cathepsin C-like protease post-translationally modifies secretory proteins for optimal invasion and egress.

Unlabelled: Microbial pathogens use proteases for their infections, such as digestion of proteins for nutrients and activation of their virulence factors. As an obligate intracellular parasite, must invade host cells to establish its intracellular propagation. To facilitate invasion, the parasites secrete invasion effectors from microneme and rhoptry, two unique organelles in apicomplexans.

Correlated Mutation in the Evolution of Catalysis in Uracil DNA Glycosylase Superfamily.

Enzymes in Uracil DNA glycosylase (UDG) superfamily are essential for the removal of uracil. Family 4 UDGa is a robust uracil DNA glycosylase that only acts on double-stranded and single-stranded uracil-containing DNA. Based on mutational, kinetic and modeling analyses, a catalytic mechanism involving leaving group stabilization by H155 in motif 2 and water coordination by N89 in motif 3 is proposed.

A structural determinant in the uracil DNA glycosylase superfamily for the removal of uracil from adenine/uracil base pairs.

The uracil DNA glycosylase superfamily consists of several distinct families. Family 2 mismatch-specific uracil DNA glycosylase (MUG) from Escherichia coli is known to exhibit glycosylase activity on three mismatched base pairs, T/U, G/U and C/U. Family 1 uracil N-glycosylase (UNG) from E.

Specificity and catalytic mechanism in family 5 uracil DNA glycosylase.

UDGb belongs to family 5 of the uracil DNA glycosylase (UDG) superfamily. Here, we report that family 5 UDGb from Thermus thermophilus HB8 is not only a uracil DNA glycosyase acting on G/U, T/U, C/U, and A/U base pairs, but also a hypoxanthine DNA glycosylase acting on G/I, T/I, and A/I base pairs and a xanthine DNA glycosylase acting on all double-stranded and single-stranded xanthine-containing DNA. Analysis of potentials of mean force indicates that the tendency of hypoxanthine base flipping follows the order of G/I > T/I, A/I > C/I, matching the trend of hypoxanthine DNA glycosylase activity observed in vitro.

The evolution of cefotaximase activity in the TEM β-lactamase.

The development of a molecular-level understanding of drug resistance through β-lactamase is critical not only in designing newer-generation antibacterial agents but also in providing insight into the evolutionary mechanisms of enzymes in general. In the present study, we have evaluated the effect of four drug resistance mutations (A42G, E104K, G238S, and M182T) on the cefotaximase activity of the TEM-1 β-lactamase. Using computational methods, including docking and molecular mechanics calculations, we have been able to correctly identify the relative order of catalytic activities associated with these four single point mutants.

New family of deamination repair enzymes in uracil-DNA glycosylase superfamily.

DNA glycosylases play a major role in the repair of deaminated DNA damage. Previous investigations identified five families within the uracil-DNA glycosylase (UDG) superfamily. All enzymes within the superfamily studied thus far exhibit uracil-DNA glycosylase activity.

The evolution of catalytic function in the HIV-1 protease.

The evolution of species is a complex phenomenon based on the optimization of a multidimensional function referred to as fitness. At the level of biomolecular evolution, the fitness function can be reduced to include physiochemical properties relevant to the biological function of a particular molecule. In this work, questions involving the physical-chemical mechanisms underlying the evolution of HIV-1 protease are addressed through molecular simulation and subsequent analysis of thermodynamic properties related to the activity of the enzyme.

Analyzing the robustness of the MM/PBSA free energy calculation method: application to DNA conformational transitions.

The ability to predict and characterize free energy differences associated with conformational equilibria or the binding of biomolecules is vital to understanding the molecular basis of many important biological functions. As biological studies focus on larger molecular complexes and properties of the genome, proteome, and interactome, the development and characterization of efficient methods for calculating free energy becomes increasingly essential. The aim of this study is to examine the robustness of the end-point free energy method termed the molecular mechanics Poisson-Boltzmann solvent accessible surface area (MM/PBSA) method.

Identification of Escherichia coli mismatch-specific uracil DNA glycosylase as a robust xanthine DNA glycosylase.

The gene for the mismatch-specific uracil DNA glycosylase (MUG) was identified in the Escherichia coli genome as a sequence homolog of the human thymine DNA glycosylase with activity against mismatched uracil base pairs. Examination of cell extracts led us to detect a previously unknown xanthine DNA glycosylase (XDG) activity in E. coli.

Thermodynamic resolution: how do errors in modeled protein structures affect binding affinity predictions?

The present study addresses the effect of structural distortion, caused by protein modeling errors, on calculated binding affinities toward small molecules. The binding affinities to a total of 300 distorted structures based on five different protein-ligand complexes were evaluated to establish a broadly applicable relationship between errors in protein structure and errors in calculated binding affinities. Relatively accurate protein models (less than 2 A RMSD within the binding site) demonstrate a 14.

Insights from xanthine and uracil DNA glycosylase activities of bacterial and human SMUG1: switching SMUG1 to UDG.

Single-strand-selective monofunctional uracil DNA glycosylase (SMUG1) belongs to Family 3 of the uracil DNA glycosylase (UDG) superfamily. Here, we report that a bacterial SMUG1 ortholog in Geobacter metallireducens (Gme) and the human SMUG1 enzyme are not only UDGs but also xanthine DNA glycosylases (XDGs). In addition, mutational analysis and molecular dynamics (MD) simulations of Gme SMUG1 identify important structural determinants in conserved motifs 1 and 2 for XDG and UDG activities.

Comparison of solvation-effect methods for the simulation of peptide interactions with a hydrophobic surface.

In this study we investigated the interaction behavior between thirteen different small peptides and a hydrophobic surface using three progressively more complex methods of representing solvation effects: a united-atom implicit solvation method [CHARMM 19 force field (C19) with Analytical Continuum Electrostatics (ACE)], an all-atom implicit solvation method (C22 with GBMV), and an all-atom explicit solvation method (C22 with TIP3P). The adsorption behavior of each peptide was characterized by the calculation of the potential of mean force as a function of peptide-surface separation distance. The results from the C22/TIP3P model suggest that hydrophobic peptides exhibit relatively strong adsorption behavior, polar and positively-charged peptides exhibit negligible to relatively weak favorable interactions with the surface, and negatively-charged peptides strongly resist adsorption.

An electrostatic basis for the stability of thermophilic proteins.

Two factors provide key contributions to the stability of thermophilic proteins relative to their mesophilic homologues: electrostatic interactions of charged residues in the folded state and the dielectric response of the folded protein. The dielectric response for proteins in a "thermophilic series" globally modulates the thermal stability of its members, with the calculated dielectric constant for the protein increasing from mesophiles to hyperthermophiles. This variability results from differences in the distribution of charged residues on the surface of the protein, in agreement with structural and genetic observations.

Native atom types for knowledge-based potentials: application to binding energy prediction.

Knowledge-based potentials have been found useful in a variety of biophysical studies of macromolecules. Recently, it has also been shown in self-consistent studies that it is possible to extract quantities consistent with pair potentials from model structural databases. In this study, we attempt to extend the results obtained from these self-consistent studies toward the extraction of realistic pair potentials from the Protein Data Bank (PDB).

Structural mining: self-consistent design on flexible protein-peptide docking and transferable binding affinity potential.

A flexible protein-peptide docking method has been designed to consider not only ligand flexibility but also the flexibility of the protein. The method is based on a Monte Carlo annealing process. Simulations with a distance root-mean-square (dRMS) virtual energy function revealed that the flexibility of protein side chains was as important as ligand flexibility for successful protein-peptide docking.

The effects of ionic strength on protein stability: the cold shock protein family.

Continuum electrostatic models are used to examine in detail the mechanism of protein stabilization and destabilization due to salt near physiological concentrations. Three wild-type cold shock proteins taken from mesophilic, thermophilic, and hyperthermophilic bacteria are studied using these methods. The model is validated by comparison with experimental data collected for these proteins.

Identifying native-like protein structures using physics-based potentials.

As the field of structural genomics matures, new methods will be required that can accurately and rapidly distinguish reliable structure predictions from those that are more dubious. We present a method based on the CHARMM gas phase implicit hydrogen force field in conjunction with a generalized Born implicit solvation term that allows one to make such discrimination. We begin by analyzing pairs of threaded structures from the EMBL database, and find that it is possible to identify the misfolded structures with over 90% accuracy.