22 research outputs found
Mid-Infrared Localized Plasmons through Structural Control of Gold and Silver Nanocrescents
Metal nanoarchitectures producing
optical responses in the visible
and near-infrared form the foundation for most plasmonic studies.
In contrast, a relative lack of infrared-active substrates has limited
the exploration of plasmonic behavior beyond the near-infrared. In
this study, we investigate the polarization-dependent, multimodal
localized plasmon resonances of asymmetric nanocrescents for large
diameter structures composed of gold and silver. The extended size
(0.5–3.0 μm) shifts the plasmon resonances into the mid-infrared
(mid-IR) spectral range. Polarization-dependent localized surface
plasmon resonance (LSPR) behavior is maintained for nanocrescent diameters
up to several microns due to the preservation of nanoscale structural
features that result in high aspect ratios. Simulations of the extinction
spectra and near-field distributions support experimentally observed
plasmonic behavior. Manipulation of nanocrescent plasmon resonances
in the mid-IR spectral range through structural-based tuning and polarization
control of incident light will find application in IR-related detection,
light guiding, and surface-enhanced IR-based spectroscopies
Characterizing the Range of Extracellular Protein Post-Translational Modifications in a Cellulose-Degrading Bacteria Using a Multiple Proteolyic Digestion/Peptide Fragmentation Approach
Post-translational modifications
(PTMs) are known to play a significant
role in many biological functions. The focus of this study is to optimize
an integrated experimental/informatics approach to more confidently
characterize the range of post-translational modifications of the
cellulosome protein complex used by the bacterium Clostridium
thermocellum to better understand how this protein
machine is tuned for enzymatic cellulose solubilization. To enhance
comprehensive characterization, the extracellular cellulosome proteins
were analyzed using multiple proteolytic digests (trypsin, Lys-C,
Glu-C) and multiple fragmentation techniques (collisionally activated
dissociation, electron transfer dissociation, decision tree). As expected,
peptide and protein identifications were increased by utilizing alternate
proteases and fragmentation methods, in addition to the increase in
protein sequence coverage. The complementarity of these experiments
also allowed for a global exploration of PTMs associated with the
cellulosome based upon a set of defined PTMs that included methylation,
oxidation, acetylation, phosphorylation, and signal peptide cleavage.
In these experiments, 85 modified peptides corresponding to 28 cellulosome
proteins were identified. Many of these modifications were located
in active cellulolytic or structural domains of the cellulosome proteins,
suggesting a level of possible regulatory control of protein function
in various cellulotyic conditions. The use of complementary proteolytic
digestion/peptide fragmentation processes allowed for independent
verification of PTMs in different experiments, thus leading to increased
confidence in PTM identifications
Characterizing the Range of Extracellular Protein Post-Translational Modifications in a Cellulose-Degrading Bacteria Using a Multiple Proteolyic Digestion/Peptide Fragmentation Approach
Post-translational modifications
(PTMs) are known to play a significant
role in many biological functions. The focus of this study is to optimize
an integrated experimental/informatics approach to more confidently
characterize the range of post-translational modifications of the
cellulosome protein complex used by the bacterium Clostridium
thermocellum to better understand how this protein
machine is tuned for enzymatic cellulose solubilization. To enhance
comprehensive characterization, the extracellular cellulosome proteins
were analyzed using multiple proteolytic digests (trypsin, Lys-C,
Glu-C) and multiple fragmentation techniques (collisionally activated
dissociation, electron transfer dissociation, decision tree). As expected,
peptide and protein identifications were increased by utilizing alternate
proteases and fragmentation methods, in addition to the increase in
protein sequence coverage. The complementarity of these experiments
also allowed for a global exploration of PTMs associated with the
cellulosome based upon a set of defined PTMs that included methylation,
oxidation, acetylation, phosphorylation, and signal peptide cleavage.
In these experiments, 85 modified peptides corresponding to 28 cellulosome
proteins were identified. Many of these modifications were located
in active cellulolytic or structural domains of the cellulosome proteins,
suggesting a level of possible regulatory control of protein function
in various cellulotyic conditions. The use of complementary proteolytic
digestion/peptide fragmentation processes allowed for independent
verification of PTMs in different experiments, thus leading to increased
confidence in PTM identifications
Characterizing the Range of Extracellular Protein Post-Translational Modifications in a Cellulose-Degrading Bacteria Using a Multiple Proteolyic Digestion/Peptide Fragmentation Approach
Post-translational modifications
(PTMs) are known to play a significant
role in many biological functions. The focus of this study is to optimize
an integrated experimental/informatics approach to more confidently
characterize the range of post-translational modifications of the
cellulosome protein complex used by the bacterium Clostridium
thermocellum to better understand how this protein
machine is tuned for enzymatic cellulose solubilization. To enhance
comprehensive characterization, the extracellular cellulosome proteins
were analyzed using multiple proteolytic digests (trypsin, Lys-C,
Glu-C) and multiple fragmentation techniques (collisionally activated
dissociation, electron transfer dissociation, decision tree). As expected,
peptide and protein identifications were increased by utilizing alternate
proteases and fragmentation methods, in addition to the increase in
protein sequence coverage. The complementarity of these experiments
also allowed for a global exploration of PTMs associated with the
cellulosome based upon a set of defined PTMs that included methylation,
oxidation, acetylation, phosphorylation, and signal peptide cleavage.
In these experiments, 85 modified peptides corresponding to 28 cellulosome
proteins were identified. Many of these modifications were located
in active cellulolytic or structural domains of the cellulosome proteins,
suggesting a level of possible regulatory control of protein function
in various cellulotyic conditions. The use of complementary proteolytic
digestion/peptide fragmentation processes allowed for independent
verification of PTMs in different experiments, thus leading to increased
confidence in PTM identifications
MOESM2 of Clostridium thermocellum DSM 1313 transcriptional responses to redox perturbation
Additional file 2. Calculated differential expression and adjusted p values for genes showing significant (adjusted p value < 0.05) differential expression during at least one timepoint of either methyl viologen or hydrogen peroxide exposure
MOESM1 of Clostridium thermocellum DSM 1313 transcriptional responses to redox perturbation
Additional file 1. Batch fermentation performance under methyl viologen and hydrogen peroxide initial loadings
MOESM3 of Clostridium thermocellum DSM 1313 transcriptional responses to redox perturbation
Additional file 3. (A) Adjusted OD600 of batch cultures grown at various initial hydrogen peroxide concentrations. Cultures were grown in MTC media containing 1.1 g/L cellobiose; (B) Chemostat OD600 and measured redox potential before, during and after hydrogen peroxide addition; (C) Detailed view of boxed region indicated in panel (B)
Development of a Multipoint Quantitation Method to Simultaneously Measure Enzymatic and Structural Components of the <i>Clostridium thermocellum</i> Cellulosome Protein Complex
<i>Clostridium thermocellum</i> has emerged as a leading
bioenergy-relevant microbe due to its ability to solubilize cellulose
into carbohydrates, mediated by multicomponent membrane-attached complexes
termed cellulosomes. To probe microbial cellulose utilization rates,
it is desirable to be able to measure the concentrations of saccharolytic
enzymes and estimate the total amount of cellulosome present on a
mass basis. Current cellulase determination methodologies involve
labor-intensive purification procedures and only allow for indirect
determination of abundance. We have developed a method using multiple
reaction monitoring (MRM-MS) to simultaneously quantitate both enzymatic
and structural components of the cellulosome protein complex in samples
ranging in complexity from purified cellulosomes to whole cell lysates,
as an alternative to a previously developed enzyme-linked immunosorbent
assay (ELISA) method of cellulosome quantitation. The precision of
the cellulosome mass concentration in technical replicates is better
than 5% relative standard deviation for all samples, indicating high
precision for determination of the mass concentration of cellulosome
components
MOESM2 of Clostridium thermocellum LL1210 pH homeostasis mechanisms informed by transcriptomics and metabolomics
Additional file 2. Raw and processed read counts, alignment statistics, log2-fold changes in the gene expression, and K-means clusters and GO enrichment of differentially expressed genes from samples taken from C. thermocellum LL1210 cultured in chemostats at pH values 6.98, 6.48, pH 6.24, and pH 6.12 (washout conditions). Gene expression at pH 6.98 was used as a reference for differential expression at lower pH values
MOESM4 of Clostridium thermocellum LL1210 pH homeostasis mechanisms informed by transcriptomics and metabolomics
Additional file 4: Figure S2. Average growth (A), terminal pH (B), and remaining substrates and products at the end of C. thermocellum-mutant strain fermentations of cellobiose in MOPS-free carbon-replete medium (C). Averages were computed with data from four biological replicates. Error bars in each graph indicate standard deviation. Some error bars are too small to see. Deletion mutants are designated as LL1210 (hydrogenase maturation protein, lactate dehydrogenase, pyruvate formate lyase, phosphotransacetylase and acetate kinase), GLDH (glutamate dehydrogenase), GS (glutamine synthetase), GOGAT (glutamate synthase), GS-GOGAT (both), and NifH (nitrogenase iron protein). The parental strain designated DSM1313 has a deletion in the hypoxanthine phosphoribosyltransferase