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The transition between dormant and active Mycobacterium tuberculosis infection requires reorganization of its lipid metabolism and activation of a battery of serine hydrolase enzymes. Among these serine hydrolases, Rv0045c is a mycobacterial-specific serine hydrolase with limited sequence homology outside mycobacteria but structural homology to divergent bacterial hydrolase families. Herein, we determined the global substrate specificity of Rv0045c against a library of fluorogenic hydrolase substrates, constructed a combined experimental and computational model for its binding pocket, and performed comprehensive substitutional analysis to develop a structural map of its binding pocket. Rv0045c showed strong substrate selectivity toward short, straight chain alkyl esters with the highest activity toward four atom chains. This strong substrate preference was maintained through the combined action of residues in a flexible loop connecting the cap and α/β hydrolase domains and in residues close to the catalytic triad. Two residues bracketing the substrate-binding pocket (Gly90 and His187) were essential to maintaining the narrow substrate selectivity of Rv0045c toward various acyl ester substituents, as independent conversion of these residues significantly increased its catalytic activity and broadened its substrate specificity. Focused saturation mutagenesis of position 187 implicated this residue, as the differentiation point between the substrate specificity of Rv0045c and the structurally homologous ybfF hydrolase family. Insertion of the analogous tyrosine residue from ybfF hydrolases into Rv0045c increased the catalytic activity of Rv0045 by over 20-fold toward diverse ester substrates. The unique binding pocket structure and selectivity of Rv0045c provide molecular indications of its biological role and evidence for expanded substrate diversity in serine hydrolases from M. tuberculosis.

Water-soluble semiconducting nanoparticles are prepared from individually collapsed and crosslinked ABA triblock copolymers and are further modified to carry imaging units and allyl functionalities for postmodification. Ethylene oxide modified polyfluorene forms the center block (B) and is transformed into a telechelic macroinitator. In a nitroxide mediated living free radical polymerization, polyacrylate blocks (A) are grown to give the ABA triblock copolymer. Low-temperature benzocyclobutene crosslinking groups are attached to collapse and site-isolate the center block (A). The nanoparticles were further modified by pegylation to enhance the solubility and by catechol groups to provide complexing sites for magnetic resonance imaging (MRI) reagents such as gadolinium. The reported materials are water-soluble and encompassing fluorescence and MRI to become biocompatible "organic quantum dots" with the possibility to interact actively with biological entities.

Serine hydrolases have diverse intracellular substrates, biological functions, and structural plasticity, and are thus important for biocatalyst design. Amongst serine hydrolases, the recently described ybfF enzyme family are promising novel biocatalysts with an unusual bifurcated substrate-binding cleft and the ability to recognize commercially relevant substrates. We characterized in detail the substrate selectivity of a novel ybfF enzyme from Vibrio cholerae (Vc-ybfF) by using a 21-member library of fluorogenic ester substrates. We assigned the roles of the two substrate-binding clefts in controlling the substrate selectivity and folded stability of Vc-ybfF by comprehensive substitution analysis. The overall substrate preference of Vc-ybfF was for short polar chains, but it retained significant activity with a range of cyclic and extended esters. This broad substrate specificity combined with the substitutional analysis demonstrates that the larger binding cleft controls the substrate specificity of Vc-ybfF. Key selectivity residues (Tyr116, Arg120, Tyr209) are also located at the larger binding pocket and control the substrate specificity profile. In the structure of ybfF the narrower binding cleft contains water molecules prepositioned for hydrolysis, but based on substitution this cleft showed only minimal contribution to catalysis. Instead, the residues surrounding the narrow binding cleft and at the entrance to the binding pocket contributed significantly to the folded stability of Vc-ybfF. The relative contributions of each cleft of the binding pocket to the catalytic activity and folded stability of Vc-ybfF provide a valuable map for designing future biocatalysts based on the ybfF scaffold., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Tularemia is a deadly, febrile disease caused by infection by the gram-negative bacterium, Francisella tularensis. Members of the ubiquitous serine hydrolase protein family are among current targets to treat diverse bacterial infections. Herein we present a structural and functional study of a novel bacterial carboxylesterase (FTT258) from F. tularensis, a homologue of human acyl protein thioesterase (hAPT1). The structure of FTT258 has been determined in multiple forms, and unexpectedly large conformational changes of a peripheral flexible loop occur in the presence of a mechanistic cyclobutanone ligand. The concomitant changes in this hydrophobic loop and the newly exposed hydrophobic substrate binding pocket suggest that the observed structural changes are essential to the biological function and catalytic activity of FTT258. Using diverse substrate libraries, site-directed mutagenesis, and liposome binding assays, we determined the importance of these structural changes to the catalytic activity and membrane binding activity of FTT258. Residues within the newly exposed hydrophobic binding pocket and within the peripheral flexible loop proved essential to the hydrolytic activity of FTT258, indicating that structural rearrangement is required for catalytic activity. Both FTT258 and hAPT1 also showed significant association with liposomes designed to mimic bacterial or human membranes, respectively, even though similar structural rearrangements for hAPT1 have not been reported. The necessity for acyl protein thioesterases to have maximal catalytic activity near the membrane surface suggests that these conformational changes in the protein may dually regulate catalytic activity and membrane association in bacterial and human homologues.

Acetyl esterases from carbohydrate esterase family 7 exhibit unusual substrate specificity. These proteins catalyze the cleavage of disparate acetate esters with high efficiency, but are unreactive to larger acyl groups. The structural basis for this distinct selectivity profile is unknown. Here, we investigate a thermostable acetyl esterase (TM0077) from Thermotoga maritima using evolutionary relationships, structural information, fluorescent kinetic measurements, and site directed mutagenesis. We measured the kinetic and structural determinants for this specificity using a diverse series of small molecule enzyme substrates, including novel fluorogenic esters. These experiments identified two hydrophobic plasticity residues (Pro228, and Ile276) surrounding the nucleophilic serine that impart this specificity of TM0077 for small, straight-chain esters. Substitution of these residues with alanine imparts broader specificity to TM0077 for the hydrolysis of longer and bulkier esters. Our results suggest the specificity of acetyl esterases have been finely tuned by evolution to catalyze the removal of acetate groups from diverse substrates, but can be modified by focused amino acid substitutions to yield enzymes capable of cleaving larger ester functionalities., (Copyright © 2012 Elsevier B.V. All rights reserved.)

In recent years, workaholism has become prevalent throughout organizations and has captured the attention of organizational leaders as well as the academic and scientific communities. Most research in this area has focused on the negative consequences of workaholism, specifically work-life imbalance. One area of research that has largely been ignored is the potential influence of demographic variables on the relationship between workaholism and work-life imbalance. Therefore, the current study focused on how cultural origin might influence the intensity of this relationship. Based on relative deprivation theory and previous empirical work, it was expected that cultural origin would moderate the relationship between workaholism and work-life imbalance. Specifically, it was predicted that Caucasian participants would score higher on levels of workaholism than Black participants, and that the relationship between workaholism and work-life imbalance would be stronger for Caucasians than for Blacks. The results revealed that high levels of workaholism were significantly correlated with high levels of work-life imbalance. However, results also indicated that cultural origin did not moderate the relationship between workaholism and work-life imbalance, and there was no significant mean difference between Caucasian and Black participants on our measure of workaholism. These findings are important in that it is essential for employers to be aware of workaholic tendencies so they can better handle the negative consequences that result for the organization, and to also help promote the well-being of their employees. En los últimos años la adicción al trabajo se ha vuelto muy popular en las organizaciones y ha capturado la atención tanto de líderes organizacionales, así como de las comunidades científicas y académicas. La mayoría de investigaciones en esta área se han focalizado en las consequencias negativas de la adicción al trabajo (workoholismo), específicamente en el desequilibrio entre trabajo-vida. Una área de investigación que ha sido ignorada por mucho tiempo es la influencia potencial de las variables demográficas sobre la relación entre adicción al trabajo y el desequilibrio trabajo-vida. Por este motivo es que el presente estudio se focalizó en como el origen cultural puede influenciar la intensidad de esta relación. Tomando como base la teoría de la deprivación relativa y estudios empíricos previos, se esperó que el origen cultural moderaría la relación entre adicción al trabajo y desequilibrio trabajo-vida. Específicamente se pudo predecir que los participantes caucásicos mostraron más elevados puntajes en los niveles de adicción al trabajo que los participantes negros, y que la relación entre adicción al trabajo y desequilibrio trabajo-vida era más intensa entre los caucásicos que entre los negros. Los resultados revelaron que elevados niveles de adicción al trabajo correlacionaron significativamente con altos niveles de desequilibrio trabajo-vida. Sin embargo los resultados también revelaron que el origen cultural no moderaba la relación entre adicción al trabajo y desequilibrio trabajo-vida, y que además no había una diferencia significativa de medias entre los participantes caucásicos y negros respecto de nuestras mediciones de adicción al trabajo. Estos resultados son importantes en la medida en que es esencial para los empleadores el ser conscientes respecto de las tendencias de adicción al trabajo, lo que les permitirá manejar con mayor efectividad las consequencias negativas para la empresa resultantes de estas tendencias, y por otro lado apoyar a sus empleados en la promoción de su bienestar. Au cours des dernières années, le travaillolisme (workaholism) a pris de l'ampleur dans les organisations et a capté l'attention à la fois des dirigeants d'organisations et des communautés académique et scientifique. La majorité de la recherche dans ce domaine fut orientée sur les conséquences négatives du travaillolisme et, plus spécifiquement, sur le conflit travail-vie. Un champ de recherche qui a été largement ignoré est l'influence potentielle des variables démographiques sur la relation entre le travaillolisme et le conflit travail-vie. La présente étude visait à comprendre comment l'origine culturelle peut influencer l'intensité de cette relation. Se basant sur la théorie de la privation relative et sur les travaux empiriques antérieurs, il était attendu que l'origine culturelle allait jouer un rôle modérateur dans la relation entre le travaillolisme et le conflit travail-vie. Plus spécifiquement, il était prédit que les participants caucasiens allaient obtenir des scores plus élevés de travaillolisme comparativement aux participants noirs, et que la relation entre le travaillolisme et le conflit travail-vie allait être plus forte pour les caucasiens que pour les noirs. Les résultats ont révélé que des niveaux élevés de travaillolisme étaient significativement corrélés avec des niveaux élevés de conflit travail-vie. Cependant, les résultats ont aussi indiqué que l'origine culturelle ne modère pas la relation entre le travaillolisme et le conflit travail-vie. De plus, il n'y avait pas de différence significative entre les participants caucasiens et les participants noirs sur notre mesure de travaillolisme. Ces résultats sont importants puisqu'il est essentiel pour les employeurs d'être à l'affût des tendances travaillolistes pour qu'ils puissent mieux en gérer les conséquences négatives pour l'organisation, ainsi que pour aider à promouvoir le bien-être de leurs employés. diferenciaban en algunos aspectos.

The controlled electrophoretic deposition of polystyrene/divinylbenzene (PS/DVB) star polymer films from a colloidal suspension is reported. Liquid suspensions, containing the PS/DVB star polymer, were prepared by injecting a dichloromethane (DCM) solution of the star polymer into a stratified liquid combination of hexane and DCM. A variety of hexane/DCM volume ratios were examined to identify the optimal solution conditions for electrophoretic deposition; thin films were produced from both unmixed and well-mixed hexane/DCM suspensions. Unmixed suspensions yielded spatially separated thin films, deposited in a controlled fashion, that were dependent on the polarity of the corresponding electrode. Films on the positive electrode differed in thickness, microstructure, and appearance from those formed on the negative electrode. In contrast, films produced from well-mixed hexane/DCM suspensions deposited uniformly across only the negative electrode. Atomic force microscopy studies revealed nanostructured surface morphologies that were unique to each of these films. Additionally, these microscopy studies shed light on the possible conformations of star polymers adsorbed on a surface. By controlling the composition and the mixing state of the solution and by controlling the bias of electrodes, we achieved controlled deposition of star polymer films with a specific nanostructure. These nanostructured films may have broad use in optical and biological device applications.