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Metabolons and supramolecular enzyme assemblies / edited by Claudia Schmidt-Dannert, Maureen B. Quin.

Contributor(s): Material type: TextTextSeries: Methods in enzymology ; v. 617.Publisher: Cambridge, MA : Academic Press, an imprint of Elsevier, 2019Edition: First editionDescription: 1 online resourceContent type:
  • text
Media type:
  • computer
Carrier type:
  • online resource
ISBN:
  • 9780128170755
  • 0128170751
Subject(s): Genre/Form: Additional physical formats: Print version:: Metabolons and supramolecular enzyme assemblies.DDC classification:
  • 660.6/34 23
LOC classification:
  • TP248.65.E59
NLM classification:
  • W1
  • QU 135
Online resources:
Contents:
Front Cover; Metabolons and Supramolecular Enzyme Assemblies; Copyright; Contents; Contributors; Preface; Chapter One: Molecular snapshots of dynamic membrane-bound metabolons; 1. Introduction; 2. SMA copolymer preparation; 2.1. SMA copolymer hydrolysis; 2.1.1. Equipment; 2.1.2. Buffers and reagents; 2.1.3. Procedures; 2.1.4. Notes; 3. Preparation of S. bicolor microsomes; 3.1. Growth of etiolated S. bicolor seedlings; 3.1.1. Equipment; 3.1.2. Buffers and reagents; 3.1.3. Procedures; 3.1.4. Notes; 3.2. Preparation of microsomes; 3.2.1. Equipment; 3.2.2. Buffers and reagents; 3.2.3. Procedures
3.2.4. Notes4. Isolation of membrane-bound metabolons from native membranes; 4.1. SMALP preparation from sorghum microsomes; 4.1.1. Equipment; 4.1.2. Buffers and reagents; 4.1.3. Procedures; 4.1.4. Notes; 4.2. Purification of SMALPs containing a target protein; 4.2.1. Equipment; 4.2.2. Buffers and reagents; 4.2.3. Procedures; 4.2.4. Notes; 5. Characterization of SMALPs containing P450 metabolons; 5.1. SMALPs observed with electron microscope; 5.1.1. Equipment; 5.1.2. Buffers and reagents; 5.1.3. Procedures; 5.1.4. Notes; 6. Summary and conclusion; References
Chapter Two: Tricarboxylic acid metabolon1. Introduction; 1.1. Importance of the TCA metabolon; 1.2. Early evidence of the TCA metabolon and challenges associated with its study; 1.3. Cross-linking mass spectrometric analysis of the TCA metabolon; 2. Preparation of cross-linked native TCA metabolon; 2.1. Isolation of intact mitochondria; 2.2. In situ cross-linking; 2.3. Isolation and characterization of metabolons; 3. Preparation of recombinant TCA metabolon; 3.1. Gene construction for recombinant TCA enzymes; 3.2. Overexpression and purification of recombinant enzymes
3.3. In-solution cross-linking and isolation of recombinant metabolons4. Mass spectrometric analysis of the Krebs cycle metabolon; 4.1. In-gel digestion of metabolons; 4.2. Mass spectrometric analysis; 4.3. Mascot database searches; 4.4. Structural reconstitution of the Krebs cycle metabolon; 5. Cross-linking techniques have allowed for the isolation of the intact metabolon; 6. Conclusions; References; Chapter Three: Metabolon formation by chemotaxis; 1. Introduction; 1.1. Metabolon formation; 1.2. Enzyme diffusion; 1.3. Enzyme chemotaxis; 2. Chemotactic assembly of enzyme cascade
2.1. Chemotactic assembly in the microfluidic channel2.1.1. Fluorescently labeling enzymes and activity assay; 2.1.2. Microfluidic device fabrication; 2.1.3. Confocal microscope imaging; 2.1.4. Chemotactic migration of hexokinase and aldolase in the microfluidic device; 2.2. Chemotactic colocalization of hexokinase and aldolase; 2.2.1. Colocalization setup; 2.2.2. Analysis of HK and Ald aggregates trajectories; 3. Summary; Acknowledgments; References; Chapter Four: Cloning, expression, and purification of intact polyketide synthase modules; 1. Introduction; 2. Cloning of intact PKS modules
Summary: Metabolons and Supramolecular Enzyme Assemblies, Volume 617 in the Methods in Enzymology series, highlights new advances in the field, with this new volume presenting interesting chapters on a variety of topics, including Dynamic plant metabolons, TCA cycle metabolons, the Chemotactic assembly of metabolons, Repurposing peroxisomes for metabolic engineering, Repurposing yeast mitochondria for metabolic engineering, Repurposing plant compartments for metabolic engineering, Protein scaffolds for pathway co-localization on lipid droplets, Engineered enzyme assemblies for metabolic engineering, NRPS assembly lines and P450 interactions, and much more. Provides the authority and expertise of leading contributors from an international board of authors Presents the latest release in this series on enzymology Updated release includes the latest information on dynamic plant metabolons, TCA cycle metabolons, the chemotactic assembly of metabolons, and much more.
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Includes bibliographical references.

Online resource; title from PDF title page (EBSCO, viewed February 26, 2019).

Front Cover; Metabolons and Supramolecular Enzyme Assemblies; Copyright; Contents; Contributors; Preface; Chapter One: Molecular snapshots of dynamic membrane-bound metabolons; 1. Introduction; 2. SMA copolymer preparation; 2.1. SMA copolymer hydrolysis; 2.1.1. Equipment; 2.1.2. Buffers and reagents; 2.1.3. Procedures; 2.1.4. Notes; 3. Preparation of S. bicolor microsomes; 3.1. Growth of etiolated S. bicolor seedlings; 3.1.1. Equipment; 3.1.2. Buffers and reagents; 3.1.3. Procedures; 3.1.4. Notes; 3.2. Preparation of microsomes; 3.2.1. Equipment; 3.2.2. Buffers and reagents; 3.2.3. Procedures

3.2.4. Notes4. Isolation of membrane-bound metabolons from native membranes; 4.1. SMALP preparation from sorghum microsomes; 4.1.1. Equipment; 4.1.2. Buffers and reagents; 4.1.3. Procedures; 4.1.4. Notes; 4.2. Purification of SMALPs containing a target protein; 4.2.1. Equipment; 4.2.2. Buffers and reagents; 4.2.3. Procedures; 4.2.4. Notes; 5. Characterization of SMALPs containing P450 metabolons; 5.1. SMALPs observed with electron microscope; 5.1.1. Equipment; 5.1.2. Buffers and reagents; 5.1.3. Procedures; 5.1.4. Notes; 6. Summary and conclusion; References

Chapter Two: Tricarboxylic acid metabolon1. Introduction; 1.1. Importance of the TCA metabolon; 1.2. Early evidence of the TCA metabolon and challenges associated with its study; 1.3. Cross-linking mass spectrometric analysis of the TCA metabolon; 2. Preparation of cross-linked native TCA metabolon; 2.1. Isolation of intact mitochondria; 2.2. In situ cross-linking; 2.3. Isolation and characterization of metabolons; 3. Preparation of recombinant TCA metabolon; 3.1. Gene construction for recombinant TCA enzymes; 3.2. Overexpression and purification of recombinant enzymes

3.3. In-solution cross-linking and isolation of recombinant metabolons4. Mass spectrometric analysis of the Krebs cycle metabolon; 4.1. In-gel digestion of metabolons; 4.2. Mass spectrometric analysis; 4.3. Mascot database searches; 4.4. Structural reconstitution of the Krebs cycle metabolon; 5. Cross-linking techniques have allowed for the isolation of the intact metabolon; 6. Conclusions; References; Chapter Three: Metabolon formation by chemotaxis; 1. Introduction; 1.1. Metabolon formation; 1.2. Enzyme diffusion; 1.3. Enzyme chemotaxis; 2. Chemotactic assembly of enzyme cascade

2.1. Chemotactic assembly in the microfluidic channel2.1.1. Fluorescently labeling enzymes and activity assay; 2.1.2. Microfluidic device fabrication; 2.1.3. Confocal microscope imaging; 2.1.4. Chemotactic migration of hexokinase and aldolase in the microfluidic device; 2.2. Chemotactic colocalization of hexokinase and aldolase; 2.2.1. Colocalization setup; 2.2.2. Analysis of HK and Ald aggregates trajectories; 3. Summary; Acknowledgments; References; Chapter Four: Cloning, expression, and purification of intact polyketide synthase modules; 1. Introduction; 2. Cloning of intact PKS modules

Metabolons and Supramolecular Enzyme Assemblies, Volume 617 in the Methods in Enzymology series, highlights new advances in the field, with this new volume presenting interesting chapters on a variety of topics, including Dynamic plant metabolons, TCA cycle metabolons, the Chemotactic assembly of metabolons, Repurposing peroxisomes for metabolic engineering, Repurposing yeast mitochondria for metabolic engineering, Repurposing plant compartments for metabolic engineering, Protein scaffolds for pathway co-localization on lipid droplets, Engineered enzyme assemblies for metabolic engineering, NRPS assembly lines and P450 interactions, and much more. Provides the authority and expertise of leading contributors from an international board of authors Presents the latest release in this series on enzymology Updated release includes the latest information on dynamic plant metabolons, TCA cycle metabolons, the chemotactic assembly of metabolons, and much more.

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