BackgroundHDX mass spectrometry is a powerful platform to probe protein structure dynamics during ligand binding, protein folding, enzyme catalysis, and such. HDX mass spectrometry analysis derives the protein structure dynamics based on the mass increase of a protein of which the backbone protons exchanged with solvent deuterium. Coupled with enzyme digestion and MS/MS analysis, HDX mass spectrometry can be used to study the regional dynamics of protein based on the m/z value or percentage of deuterium incorporation for the digested peptides in the HDX experiments. Various software packages have been developed to analyze HDX mass spectrometry data. Despite the progresses, proper and explicit statistical treatment is still lacking in most of the current HDX mass spectrometry software. In order to address this issue, we have developed the HDXanalyzer for the statistical analysis of HDX mass spectrometry data using R, Python, and RPY2.Implementation and resultsHDXanalyzer package contains three major modules, the data processing module, the statistical analysis module, and the user interface. RPY2 is employed to enable the connection of these three components, where the data processing module is implemented using Python and the statistical analysis module is implemented with R. RPY2 creates a low-level interface for R and allows the effective integration of statistical module for data processing. The data processing module generates the centroid for the peptides in form of m/z value, and the differences of centroids between the peptides derived from apo and ligand-bound protein allow us to evaluate whether the regions have significant changes in structure dynamics or not. Another option of the software is to calculate the deuterium incorporation rate for the comparison. The two types of statistical analyses are Paired Student’s t-test and the linear combination of the intercept for multiple regression and ANCOVA model. The user interface is implemented with wxpython to facilitate the data visualization in graphs and the statistical analysis output presentation. In order to evaluate the software, a previously published xylanase HDX mass spectrometry analysis dataset is processed and presented. The results from the different statistical analysis methods are compared and shown to be similar. The statistical analysis results are overlaid with the three dimensional structure of the protein to highlight the regional structure dynamics changes in the xylanase enzyme.ConclusionStatistical analysis provides crucial evaluation of whether a protein region is significantly protected or unprotected during the HDX mass spectrometry studies. Although there are several other available software programs to process HDX experimental data, HDXanalyzer is the first software program to offer multiple statistical methods to evaluate the changes in protein structure dynamics based on HDX mass spectrometry analysis. Moreover, the statistical analysis can be carried out for both m/z value and deuterium incorporation rate. In addition, the s...
Twenty-three populations belonging to 10 wild Primula L. (Primulaceae) taxa of Turkey, some of which are morphologically quite similar, were investigated based on nrDNA ITS regions. The plant materials were collected from different geographical areas of Anatolia in vegetation periods of 2005 and 2006. Total genomic DNAs were isolated from the healthy leaves of each population. The entire ITS regions of the populations were amplified by universal primers with the aid of a polymerase chain reaction (PCR), and then the PCR products were sequenced. Neighbour-Joining (NJ) and Maximum Parsimony (MP) trees were constructed in order to identify the relationships among Primula taxa. According to ITS data, 724 characters were determined among the aligned sequences of the populations and the divergence values were found to be between 0.0% and 20.9%. ITS sequences from 23 specimens provided a number of variable and sufficient characters to explore the relationships. As a result, it was determined that the dendrograms obtained by NJ and MP analysis are concordant with the traditional taxonomical order at subgeneric level.
Endocrine disrupters include a broad spectrum of chemicals such as industrial chemicals, natural estrogens and androgens, synthetic estrogens and androgens. Phytoestrogens are widely present in diet and food supplements; mycoestrogens are frequently found in grains. As human beings and animals are commonly exposed to phytoestrogens and mycoestrogens in diet and environment, it is important to understand the potential beneficial or hazardous effects of estrogenic compounds. Many bioassays have been established to study the binding of estrogenic compounds with estrogen receptor (ER) and provided rich data in the literature. However, limited assays can offer structure information with regard to the ligand/ER complex. Our current study surveys the global structure dynamics changes for ERα ligand binding domain (LBD) when phytoestrogens and mycoestrogens bind. The assay is based on the structure dynamics information probed by hydrogen deuterium exchange mass spectrometry and offers a unique viewpoint to elucidate the mechanism how phytoestrogens and mycoestrogens interact with estrogen receptor. The cluster analysis based on the hydrogen deuterium exchange (HDX) assay data reveals a unique pattern when phytoestrogens and mycoestrogens bind with ERα LBD compared to that of estradiol and synthetic estrogen modulators. Our study highlights that structure dynamics could play an important role in the structure function relationship when endocrine disrupters interact with estrogen receptors.
Background: Insect gut symbionts are essential for biomass degradation, nutrient synthesis and toxin detoxification. We carried out comparative ana lysis of gut symbionts from four insect species to investigate the adaptation to different food types and to evaluate the potential for biofuel applications. Results: The study revealed significant variation of insect gut symbiont composition as a function to adapt to different food types. The composition-function relationship has been discussed from different perspectives, including biomass utilization and nutrient biosynthesis. The existence of species such as Klebsiella oxytoca and the capacity for these species to produce unique biomass degradation enzymes indicated that grasshopper and woodborer gut systems may be enriched resources for biofuel applications. For functional verification and concept validation, we have cloned and characterized a b-glucosidase from K. oxytoca in the grasshopper gut system. Conclusion: The research suggested that different insect gut systems can be good resources for biocatalyst and microbial strain discovery for bioenergy applications.
BackgroundEnzyme dynamics has recently been shown to be crucial for structure-function relationship. Among various structure dynamics analysis platforms, HDX (hydrogen deuterium exchange) mass spectrometry stands out as an efficient and high-throughput way to analyze protein dynamics upon ligand binding. Despite the potential, limited research has employed the HDX mass spec platform to probe regional structure dynamics of enzymes. In particular, the technique has never been used for analyzing cell wall degrading enzymes. We hereby used xylanase as a model to explore the potential of HDX mass spectrometry for studying cell wall degrading enzymes.ResultsHDX mass spectrometry revealed significant intrinsic dynamics for the xylanase enzyme. Different regions of the enzymes are differentially stabilized in the apo enzyme. The comparison of substrate-binding enzymes revealed that xylohexaose can significantly stabilize the enzyme. Several regions including those near the reaction centres were significantly stabilized during the xylohexaose binding. As compared to xylohexaose, xylan induced relatively less protection in the enzyme, which may be due to the insolubility of the substrate. The structure relevance of the enzyme dynamics was discussed with reference to the three dimensional structure of the enzyme. HDX mass spectrometry revealed strong dynamics-function relevance and such relevance can be explored for the future enzyme improvement.ConclusionLigand-binding can lead to the significant stabilization at both regional and global level for enzymes like xylanase. HDX mass spectrometry is a powerful high-throughput platform to identify the key regions protected during the ligand binding and to explore the molecular mechanisms of the enzyme function. The HDX mass spectrometry analysis of cell wall degrading enzymes has provided a novel platform to guide the rational design of enzymes.
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