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Identifying and characterizing the enzymes responsible for an observed activity within a complex eukaryotic catabolic system remains one of the most significant challenges in the study of biomass-degrading systems. The debranching of both complex hemicellulosic and pectinaceous polysaccharides requires the production of α-L-arabinofuranosidases among a wide variety of co-expressed carbohydrate-active enzymes. To selectively detect and identify α-L-arabinofuranosidases produced by fungi grown on complex biomass, potential covalent inhibitors and probes which mimic α-L-arabinofuranosides were sought. The conformational free energy landscapes of free α-L-arabinofuranose and several rationally designed covalent α-L-arabinofuranosidase inhibitors were analyzed. A synthetic route to these inhibitors was subsequently developed based on a key Wittig-Still rearrangement. Through a combination of kinetic measurements, intact mass spectrometry, and structural experiments, the designed inhibitors were shown to efficiently label the catalytic nucleophiles of retaining GH51 and GH54 α-L-arabinofuranosidases. Activity-based probes elaborated from an inhibitor with an aziridine warhead were applied to the identification and characterization of α-L-arabinofuranosidases within the secretome of A. niger grown on arabinan. This method was extended to the detection and identification of α-L-arabinofuranosidases produced by eight biomass-degrading basidiomycete fungi grown on complex biomass. The broad applicability of the cyclophellitol-derived activity-based probes and inhibitors presented here make them a valuable new tool in the characterization of complex eukaryotic carbohydrate-degrading systems and in the high-throughput discovery of α-L-arabinofuranosidases.
This article was published in the following journal.
Name: Journal of the American Chemical Society
Clan CA cysteine proteases, also known as papain-like proteases, play important roles throughout the malaria parasite life cycle and are therefore potential drug targets to treat this disease and prev...
Activity-based protein profiling (ABPP) is a robust chemoproteomic technique that uses activity-based probes to globally measure endogenous enzymatic activity in complex proteomes. It has been utilize...
Development of next-generation fluorescent probes is a key element in the quest for greater understanding of complex biological environments (e.g. membranes) by bioimaging. Such fluorescence-based tec...
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Bioorthogonal chemistry allows the selective modification of biomolecules in complex biological samples. One application of this methodology is in two-step activity-based protein profiling (ABPP), a m...
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This observational study evaluates if drug response testing can be performed within 7 days and analyzes the value of ex-vivo drug screening for hematological malignancies as a biomarker to...
The findings of this study will advance movement reorganization mechanism underlying treatment approaches and clinical intervention techniques. These findings may inform rehabilitation pro...
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A non-randomised mixed methods controlled before-and-after study. The study evaluated the effects of a classroom-based physical activity intervention in primary school children on physical...
An approach, process, or methodology which emphasizes credible evidence and the best available scientific knowledge, judiciously integrated to achieve the best possible outcomes in structural design. For example, the design of a new OUTPATIENT CLINIC might incorporate a review of published research on outpatient clinic design, decisions on similar past projects, along with interviews with staff and consumers.
A DNA amplification technique based upon the ligation of OLIGONUCLEOTIDE PROBES. The probes are designed to exactly match two adjacent sequences of a specific target DNA. The chain reaction is repeated in three steps in the presence of excess probe: (1) heat denaturation of double-stranded DNA, (2) annealing of probes to target DNA, and (3) joining of the probes by thermostable DNA ligase. After the reaction is repeated for 20-30 cycles the production of ligated probe is measured.
The molecular designing of drugs for specific purposes (such as DNA-binding, enzyme inhibition, anti-cancer efficacy, etc.) based on knowledge of molecular properties such as activity of functional groups, molecular geometry, and electronic structure, and also on information cataloged on analogous molecules. Drug design is generally computer-assisted molecular modeling and does not include pharmacokinetics, dosage analysis, or drug administration analysis.
The degree of 3-dimensional shape similarity between proteins. It can be an indication of distant AMINO ACID SEQUENCE HOMOLOGY and used for rational DRUG DESIGN.
RNA, usually prepared by transcription from cloned DNA, which complements a specific mRNA or DNA and is generally used for studies of virus genes, distribution of specific RNA in tissues and cells, integration of viral DNA into genomes, transcription, etc. Whereas DNA PROBES are preferred for use at a more macroscopic level for detection of the presence of DNA/RNA from specific species or subspecies, RNA probes are preferred for genetic studies. Conventional labels for the RNA probe include radioisotope labels 32P and 125I and the chemical label biotin. RNA probes may be further divided by category into plus-sense RNA probes, minus-sense RNA probes, and antisense RNA probes.