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NMR-Bioassay Guided Isolation of the Natural 20S Proteasome Inhibitors from Photorhabdus Luminescens : A Novel NMR-Tool for Natural Product Detection / by Martin Lorenz Stein
Resource type: Ressourcentyp: Buch (Online)Book (Online)Language: English Series: Springer Theses, Recognizing Outstanding Ph.D. Research | SpringerLink BücherPublisher: Cham ; s.l. : Springer International Publishing, 2014Description: Online-Ressource (XVI, 71 p. 28 illus., 24 illus. in color, online resource)ISBN:- 9783319079141
- 543.2543.8
- 543.0877 23
- QD95-96
Contents:
Summary: Martin Stein's thesis describes a novel methodology for natural product discovery. Due to its high degree of reproducibility, robustness and sensitivity, the technique can be utilized to detect even trace amounts of bioactive substances in heterogeneous matrices such as fermentation broths or crude organic extracts. This research is thus relevant for a large number of researchers working in natural product discovery. Applications of this novel NMR-based approach include suitable environmental triggers for the induction of biosynthetic machineries. The author demonstrates the extraordinary value of this approach by the successful isolation of two potent inhibitors of the pharmaceutically relevant proteasome core particle from the insect pathogen photorhabdus luminescens. This thesis has led to a number of publications in high-impact journalsPPN: PPN: 1658627970Package identifier: Produktsigel: ZDB-2-CMS
Supervisor's Foreword; Acknowledgments; Contents; Abbreviations; 1 Introduction; 1.1 The Ubiquitin-Proteasome-System; 1.1.1 Ubiquitinylation; 1.1.2 The 26S Proteasome; 1.1.3 The Core Particle; 1.2 Principles and Applications of Synthetic Proteasome Inhibitors; 1.3 Natural Proteasome Inhibitors; 1.4 Proteasome Inhibitors as Virulence Factors from Pathogenic Origin; 1.5 Overview of Currently Applied Methods for Proteasomal Inhibitor Detection; References; 2 Objective; 3 Materials and Methods; 3.1 Materials; 3.1.1 Chemicals; 3.1.2 Growth Media; 3.1.3 Strains; 3.1.4 Fluorogenic Substrates
3.1.5 Instruments3.1.6 Computer Software and Bioinformatic Tools; 3.2 Generation of Peptide Substrates; 3.2.1 Solid Phase Synthesis; 3.2.2 Resin Separation and Purification; 3.3 Protein Chemistry; 3.3.1 Purification of the 20S Proteasome; 3.3.2 SDS-PAGE Analysis; 3.3.3 Determination of Protein Concentrations; 3.4 Activity Assays; 3.4.1 Artificial Fluorescent Probes and IC50 Measurements; 3.4.2 Native Gel Fluorescence Analysis; 3.4.3 SDS-PAGE Shift Assay; 3.4.4 Peptide Digestion and Product Cleavage Pattern Analysis; 3.4.5 NMR Analysis of Labeled Peptide Substrates
3.5 Isolation of Natural Products3.5.1 Screening of Growth Conditions; 3.5.2 Secretion and Purification of the Syrbactins CepI and GlbA; 3.5.3 LC-MS, 1D- and 2D-NMR Structure Elucidation; 3.6 Protein Crystallography; 3.6.1 Crystallization of the Yeast 20S Proteasome; 3.6.2 Data Collection, Processing and Structure Determination; 3.7 Cell Culture Experiments; 3.7.1 Cell Viability Assays and Kinetic Experiments; 3.7.2 NF-κB Pathway Analysis; References; 4 Results; 4.1 Development of a NMR-Based Proteasome Assay; 4.1.1 Peptide Design and Digestion Analyses
4.1.2 NMR Analysis of the Cleavage Reaction4.1.3 Primary Sequence Optimization; 4.2 Assay Verification in a Real-Case Scenario; 4.2.1 Detection of SylA in Cultures of P. syringae by Orthogonal Methodologies; 4.2.2 Analysis of SylA Concentrations by NMR Assays; 4.3 Induction of Proteasome Inhibitor Secretion in P. luminescens; 4.3.1 Triggering the Pathogenic Phase in Photorhabdus; 4.3.2 Isolation and Structure Elucidation of the Natural Proteasome Inhibitors; 4.4 Functional and Structural Characterization of CepI and GlbA; 4.4.1 Structure Elucidation by 2D-NMR Spectroscopy
4.4.2 Reversibility Assays, Selectivity and IC50 Measurements4.4.3 Analysis of the Yeast 20S Proteasome: Ligand Complex Structures; 4.4.4 Target Specific Adaptations of Syrbactins; 4.5 Cell Viability and Pathway Specific Protein Accumulation Assays; 4.5.1 Cell Cytotoxicity Measurements; 4.5.2 Pathway Selective Accumulation Assay; References; 5 Discussion; 5.1 Development of a Tool for the Identification of CP Inhibitors from Natural Sources; 5.2 Isolation and Characterization of CepI and GlbA; References
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