is the causative agent of the highly, infectious disease, tularemia. Amongst the genes identified as essential to the virulence of
was the proposed serine hydrolase FTT0941c. Herein, we purified ...FTT0941c to homogeneity and then characterized the folded stability, enzymatic activity, and substrate specificity of FTT0941c. Based on phylogenetic analysis, FTT0941c was classified within a divergent
subbranch of the bacterial hormone sensitive lipase (HSL) superfamily, but with the conserved sequence motifs of a bacterial serine hydrolase. FTT0941c showed broad hydrolase activity against diverse libraries of ester substrates, including significant hydrolytic activity across alkyl ester substrates from 2 to 8 carbons in length. Among a diverse library of fluorogenic substrates, FTT0941c preferred α-cyclohexyl ester substrates, matching with the substrate specificity of structural homologues and the broad open architecture of its modeled binding pocket. By substitutional analysis, FTT0941c was confirmed to have a classic catalytic triad of Ser115, His278, and Asp248 and to remain thermally stable even after substitution. Its overall substrate specificity profile, divergent phylogenetic homology, and preliminary pathway analysis suggested potential biological functions for FTT0941c in diverse metabolic degradation pathways in
.
Abstract only
The serine hydrolase FTT258 from the highly pathogenic bacterium
Francisella tularensis
exists in distinct open and closed conformations. Based on comparison to the homologous human ...acyl protein thioesterase, this large structural rearrangement was proposed to provide connected control over the catalytic and membrane binding activity of FTT258. Using comprehensive mutagenesis and differential kinetic and membrane‐binding measurements, we determined the contribution of a key flexible loop to controlling the structural rearrangement and biological activity of FTT258. For the catalytic activity, a centrally located tryptophan residue (Trp66) was deemed essential, with the resulting alanine variant showing complete ablation of enzyme activity. Other amino acids localized near this essential tryptophan residue, including Met63, Arg64, Tyr67, and Asp68, also significantly decreased the hydrolase activity, indicating a critical target area for controlling the enzyme activity and structural rearrangement of FTT258. Combinatorial variants containing the Trp66 substitution with other important residues showed enzymatic activity similar to the Trp66 variant alone, suggesting a controlling role for this residue. Liposome‐binding experiments with the tryptophan variants did not however show significant changes in membrane binding activity. Instead, removal of a proximal positively‐charged arginine residue (Arg64) significantly decreased the membrane binding activity of FTT258. Together, the conformational change in FTT258 dually controlled the catalytic and membrane binding activity of FTT258, but through distinct subsections of a key flexible loop.
Support or Funding Information
Funding provided by a Butler University Holcomb research award and a Senior Research Grant from the Indiana Academy of Sciences.
Metastable Atom De-excitation Spectroscopy (MDS) provides a powerful technique with which to investigate surface electronic structure with unparalleled surface specificity. In this technique a ...thermal energy beam of noble-gas metastable atoms is directed at the surface under study and the kinetic energy distribution of ejected electrons that result from metastable atom de-excitation is measured. Although the measured distribution contains information about the electronic structure of the outermost surface layer, its detailed analysis requires knowledge of the dynamics of the metastable atom-surface interaction. In the present work, these dynamics have been investigated directly, for the first time, by use of spin-labeling techniques. The electron spins on the incident metastable atoms are polarized and the spin-polarization of the ejected electrons is measured with a Mott polarimeter. Results are reported for Cu(100) and Ni(111) surfaces under a variety of surface conditions. The data indicate that metastable atom-surface interactions may be more complex than is generally assumed and thus suggest that assumptions inherent in earlier analyses of MDS spectra merit additional investigation. The present work further demonstrates the power of spin-resolved measurements in the study of particle-surface interactions and in surface spectroscopies in general.
Metastable Atom De-excitation Spectroscopy (MDS) provides a powerful technique with which to investigate surface electronic structure with unparalleled surface specificity. In this technique a ...thermal energy beam of noble-gas metastable atoms is directed at the surface under study and the kinetic energy distribution of ejected electrons that result from metastable atom de-excitation is measured. Although the measured distribution contains information about the electronic structure of the outermost surface layer, its detailed analysis requires knowledge of the dynamics of the metastable atom-surface interaction. In the present work, these dynamics have been investigated directly, for the first time, by use of spin-labeling techniques. The electron spins on the incident metastable atoms are polarized and the spin-polarization of the ejected electrons is measured with a Mott polarimeter. Results are reported for Cu(100) and Ni(111) surfaces under a variety of surface conditions. The data indicate that metastable atom-surface interactions may be more complex than is generally assumed and thus suggest that assumptions inherent in earlier analyses of MDS spectra merit additional investigation. The present work further demonstrates the power of spin-resolved measurements in the study of particle-surface interactions and in surface spectroscopies in general.
The primary muscle disorders are a diverse group of diseases caused by various defective structural proteins, abnormal signaling molecules, enzymes and proteins involved in posttranslational ...modifications, and other mechanisms. Although there is increasing clarification of the primary aberrant cellular processes responsible for these conditions, the decisive factors involved in the secondary pathogenic cascades are still mainly obscure. Given the emerging roles of microRNAs (miRNAs) in modulation of cellular phenotypes, we searched for miRNAs regulated during the degenerative process of muscle to gain insight into the specific regulation of genes that are disrupted in pathological muscle conditions. We describe 185 miRNAs that are up- or down-regulated in 10 major muscular disorders in humans Duchenne muscular dystrophy (DMD), Becker muscular dystrophy, facioscapulohumeral muscular dystrophy, limb-girdle muscular dystrophies types 2A and 2B, Miyoshi myopathy, nemaline myopathy, polymyositis, dermatomyositis, and inclusion body myositis. Although five miRNAs were found to be consistently regulated in almost all samples analyzed, pointing to possible involvement of a common regulatory mechanism, others were dysregulated only in one disease and not at all in the other disorders. Functional correlation between the predicted targets of these miRNAs and mRNA expression demonstrated tight posttranscriptional regulation at the mRNA level in DMD and Miyoshi myopathy. Together with direct mRNA-miRNA predicted interactions demonstrated in DMD, some of which are involved in known secondary response functions and others that are involved in muscle regeneration, these findings suggest an important role of miRNAs in specific physiological pathways underlying the disease pathology.
Relationships between shifts in climatic and other environmental conditions and changes in the character and dynamics of phytoplankton blooms were examined in three interconnected subtropical lagoons ...on the east coast of Florida, i.e., Mosquito Lagoon, Indian River Lagoon, and Banana River Lagoon, from 1997 to 2013. Phytoplankton blooms were a common feature through most of the study period in two of the lagoons. From 1997 to 2009, blooms in the latter two lagoons were typically dominated by dinoflagellates in the warm wet season and diatoms in the cool dry season. Blooms of the dominant bloom-forming dinoflagellate species Pyrodinium bahamense were positively correlated to rainfall levels, indicating a link to enhanced external nutrient loads. In 2011–2013, major blooms were observed in all three lagoons, but unlike the previous 14 years, they were dominated by picoplanktonic eukaryotes, including a chlorophyte, Pedinophyceae sp., and the brown tide species Aureoumbra lagunensis. The results suggest that extreme climatic conditions, including record cold winter water temperatures and low rainfall levels, were major driving factors in this state shift in the character of blooms, through a wide range of effects including die-offs of benthic flora and fauna, suppression of grazer populations, alteration of nutrient regimes, and uncharacteristic water column conditions, such as elevated salinities and light attenuation.
Nurse sharks have not previously been known to migrate. Nurse sharks of the Dry Tortugas (DRTO) mating population have a highly predictable periodic residency cycle, returning to the Dry Tortugas ...Courtship and Mating Ground (DTCMG) annually (males) or bi- to triennially (females) during the June/July mating season. For 23 years we have followed the movements of 76 recaptured adults of a total of 115 tagged adults. Telemetry detections of 40 females tagged with acoustic transmitters show that most tagged and presumably post-partum females are continuously present in the DRTO in the fall, winter and early spring following the June mating season but these females depart in late March to early May. Detections reveal these females avoid the DTCMG completely during the next mating season, returning from late summer to fall. Telemetry records of nine of 17 adult males that co-habited with these females in the DTCMG depart DRTO waters every July. Both sexes may overwinter in the DRTO. Between 2011 and 2016 three males and five females with transmitters were detected to move up the west coast of Florida outside of the mating season as far north as the waters off Tampa Bay (335 km). Six others were only detected in the lower Florida Keys (292 km). Nine sharks returned to DRTO; one returned six times. Some overwintered and some resumed courtship in June, demonstrating both resident and migratory contingents within their population, partial migration and an ability to navigate with high spatial and temporal precision.
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