Fe-S cluster enzymes. Part B / edited by Sheila S. David.

Format:

Book

Contributor:

David, Sheila S., editor.

Series:

Methods in enzymology ; Volume 599.

Methods in Enzymology ; Volume 599

Language:

English

Subjects (All):

Iron-sulfur proteins.

Physical Description:

1 online resource (491 pages) : illustrations.

Place of Publication:

Cambridge, Massachusetts : Academic Press, 2018.

Summary:

Methods in Enzymology, Volume 599 is the second of two volumes focused on Fe-S cluster enzymes. Topics of interest in this new release include steps towards understanding mitochondrial Fe/S cluster biogenesis, iron sulfur clusters in zinc finger proteins, electrochemistry of Iron-sulfur enzymes, NRVS for Fe in biology and its experiment and basic interpretation,methods for studying iron regulatory protein 1, an important protein in human iron metabolism, the characterization of glutaredoxin Fe-S cluster binding interactions using circular dichroism spectroscopy, fluorescent reporters to track Fe-S cluster assembly and transfer reactions, methods for studying the Fe-S cluster containing base excision repair glycosylase MUTYH, and more.- Contain contributions from leading authorities on enzymology- Informs and updates on all the latest developments in the field

Contents:

Front Cover

Fe-S Cluster Enzymes Part B

Copyright

Contents

Contributors

Preface

Chapter One: Iron-Sulfur Clusters in DNA Polymerases and Primases of Eukaryotes

1. Introduction

2. Iron-Sulfur Clusters in DNA Polymerases and Primase

2.1. DNA Polymerases at the Replication Fork

2.2. Iron-Sulfur Cluster in the Large Subunit of DNA Primase

2.3. Iron-Sulfur Clusters in B-Family DNA Polymerases

3. Genetic Evidence for the Important Roles of Iron-Sulfur Clusters in DNA Replication In Vivo

4. Purification of Eukaryotic B-Family DNA Polymerases

4.1. Procedure

4.2. Notes

5. Analysis of Iron Content in Protein Samples

5.1. Procedure

5.2. Notes

Acknowledgment

References

Chapter Two: Fe-S Clusters and MutY Base Excision Repair Glycosylases: Purification, Kinetics, and DNA Affinity Measurements

2. Over Expression and Purification of MutY Homologs

2.1. Considerations for Isolating MutY Homologs

2.2. Bacteria as an Overexpression Host

2.3. Purification of E. coli MutY

2.4. Purification of a Thermophilic Homolog, G. stearothermophilus MutY

2.5. MBP-MutY for Higher Yields and Solubility

2.6. Bacterial Expression of M. musculus Mutyh

2.7. Eukaryotic Expression System for Production of Homo sapiens MUTYH

3. Gel-Based Adenine Glycosylase Assays and Measurements of Kinetic Parameters

3.1. General Setup and Execution of the Glycosylase Assay

3.1.1. Radiolabeling the DNA Substrate

3.1.2. Preparation of a Denaturing Polyacrylamide Gel

3.1.3. General Assay Setup

3.1.4. Visualization and Quantitation of Results

3.1.5. Salt Concentration in Assay Buffer

3.2. Correcting the Enzyme Concentration for the Percent Active Fraction

3.3. Determining the Rate of Product Release

3.4. Assessing the Rate of Glycosidic Bond Cleavage.

4. Gel-Based Assays for Determining MutY-DNA Affinity

4.1. General Features and Considerations of Gel-Based Binding Assays With MutY

4.1.1. Radiolabeling the DNA Substrate

4.1.2. Preparation of a Nondenaturing Polyacrylamide Gel

4.1.3. Running an EMSA

4.1.4. Visualization and Quantitation of Results

4.2. Measurements of MutY-DNA Dissociation Constants

4.3. Measurement of DNA Dissociation Rate (koff)

5. Application of Methods to Reveal Roles of the Fe-S Cluster Cofactor in MutY Homologs

Acknowledgments

Chapter Three: Cellular Assays for Studying the Fe-S Cluster Containing Base Excision Repair Glycosylase MUTYH and Homologs

2. Mutation Suppression Activity Measured in Rifampicin Resistance Assays

2.1. Overview of the Rifampicin Resistance Assay

2.2. Transformation and Growth of Cells

2.3. Determining the Mutation Frequency

2.3.1. Troubleshooting Tips

2.4. Case Study: Use of Rifampicin Resistance Assay to Characterize Zn-Linchpin Motif in MUTYH

3. Analysis of MutY-Mediated Repair of Defined Plasmid Substrates in E. coli

3.1. Designing a Plasmid-Based Bacterial Cell Assay

3.2. Selection of Cloning Vector and Design of Insertion Sequence

3.3. Electrocompetency and Electroporation Protocol

3.4. Amplification and Plasmid Extraction

3.5. Agarose Gel and Confirmation by Sequencing

3.5.1. Troubleshooting Tips

3.6. Case Study: Use of Bacterial OG:A Repair Assay to Assess Relative Impacts of Catalytic vs Binding Defects in MutY

4. A Mammalian Cell-Based GFP Reporter Assay

4.1. Evaluating DNA Repair in a Mammalian Cell-Based System

4.2. Design of Mammalian Plasmid Reporter Constructs

4.3. Production of Plasmid Reporter Constructs

4.3.1. Troubleshooting Tips

4.4. Quantification of Expression With Competitive RT-PCR.

4.5. Measuring Repair With a GFP Reporter

4.5.1. Troubleshooting Tips

4.6. Case Study: Evaluating MAP Variants in Mammalian Cells

5. Summary and Conclusion

Chapter Four: Iron-Sulfur Clusters in Zinc Finger Proteins

2. Approaches to Clone Zinc Finger/Fe-S Cluster Genes

2.1. Cloning Strategy

2.1.1. Equipment

2.1.2. Buffers and Reagents

2.1.3. Protocol

3. Expression of ZF Proteins and Adaptations for Inclusion of Iron-Sulfur Clusters

3.1. General Protocol for Expression of Zinc Finger Proteins Containing Iron-Sulfur Clusters

3.1.1. Equipment

3.1.2. Buffers and Reagents for LB Overexpression

3.1.3. Protocol

3.2. Cell Lysis

3.2.1. Equipment

3.2.2. Buffers and Reagents

3.2.3. General Sonication Protocol

4. Protein Purification

4.1. Amylose Column Chromatography

4.1.1. Equipment

4.1.2. Buffers and Reagents

4.1.3. General Amylose Affinity Column Chromatography Purification Protocol

4.2. Additional Polishing Step via Size Exclusion Chromatography

5. Methods to Characterize ZF Proteins With Fe-S Clusters

5.1. Protein Characterization Using UV-vis

5.1.1. Equipment

5.1.2. Buffers and Reagents

5.1.3. General UV-vis Characterization Protocol for Proteins Containing Zinc Finger and Iron-Sulfur Clusters

5.2. ICP-MS

5.3. XAS Sample Preparation

6. Activity Assays to Assess DNA or RNA Binding for ZF/Fe-S Hybrid Proteins

6.1. Evaluation of CPSF30/RNA Binding via EMSA

6.2. Quantification of ZF/RNA Binding via Fluorescence Anisotropy

7. Conclusions

Chapter Five: Methods for Studying Iron Regulatory Protein 1: An Important Protein in Human Iron Metabolism

2. Aconitase Activity Assays

2.1. Spectrophotometric Aconitase Assay.

2.2. In-Gel Aconitase Activity Assay

3. Electrophoretic Mobility Shift Assay

4. Targets of IRP1

5. Reconstitution, Bioanalytical, and Biophysical Analysis of IRP1

6. Conclusions

Chapter Six: Robust Production, Crystallization, Structure Determination, and Analysis of [Fe-S] Proteins: Uncovering Con ...

2. Recombinant [Fe-S] Protein Production and Purification in General: What Is Needed and What Needs to Be Considered?

2.1. [Fe-S] Protein Production

2.2. [Fe-S] Protein Purification: Use Anaerobic Hood and Check for Full Fe-S Occupancy

3. [Fe-S] Protein Crystallization, Data Collection, and Structure Determination

3.1. Considerations and Strategies for Anaerobic Crystallization

3.2. Protocols for Anaerobic Crystal Mounting

3.3. Data Collection Strategy for [Fe-S] Proteins Employing Fe Phasing With Minimum Exposure and Maximum Redundancy

3.4. Structure Determination: Proper Refinement to Maintain Cluster Geometry and Occupancy

4. Structural Analyses: What to Look for in Fe-S Cluster Structures: Case Studies in Type II Mo-bisMGD Enzymes

5. Summary and Prospects for Advances

Chapter Seven: Biochemical Reconstitution and Spectroscopic Analysis of Iron-Sulfur Proteins

1.1. Fe/S Clusters and Their Coordination

1.2. The Mitochondrial ISC Machinery as a Paradigm Fe/S Protein Biogenesis System

2. Chemical and Biochemical Reconstitution of Simple Fe/S Proteins

2.1. Equipment

2.2. Buffers and Reagents

2.3. Procedures

2.3.1. Chemical Reconstitution

2.3.2. Semienzymatic Reconstitution

2.4. Notes

3. De Novo Synthesis of a [2Fe-2S] Clusters on the Scaffold Protein Isu1 In Vitro

3.1. Equipment

3.2. Buffers and Reagents

3.3. Procedures

3.4. Notes.

4. At a Glance: Spectroscopic Characterization of Fe/S Proteins

4.1. Optical Absorption and CD Spectroscopy

4.2. EPR Spectroscopy

4.3. Mössbauer Spectroscopy

4.4. Equipment

4.5. Procedures

4.5.1. Sample Preparation for EPR

4.5.2. Sample Preparation for Mössbauer

Chapter Eight: Biochemical Analyses of Human Iron-Sulfur Protein Biogenesis and of Related Diseases

2. Analysis of Fe/S Protein Assembly in Tissue Material by Immunoblotting

2.1. Digitonin-Based Fractionation of Patient-Derived and Other Cultured Human Cells

2.1.3. Procedure

2.1.4. Notes

2.2. Preparation of Cell Extracts From Skeletal Muscle Biopsies

2.2.1. Equipment

2.2.2. Buffers and Reagents

2.2.3. Procedure

2.2.4. Notes

2.3. TCA Precipitation of Proteins for Subsequent SDS-PAGE

2.3.1. Equipment

2.3.2. Buffers and Reagents

2.3.3. Procedure

2.3.4. Notes

2.4. Preparation of Gradient Gels for SDS-PAGE-Based Separation of Cellular Proteins

2.4.1. Equipment

2.4.2. Buffers and Reagents

2.4.3. Procedure

2.4.4. Notes

3. Strategies to Assay Iron-Sulfur Protein Assembly Defects by Immunoblotting

3.1. Assessing Fe/S Protein Steady-State Levels

3.1.1. Analysis of Defects in Mitochondrial Core ISC Components

3.1.2. Analysis of Defects in Late-Acting Mitochondrial ISC-Targeting Factors

3.1.3. Analysis of Defects in Cytosolic-Nuclear Fe/S Protein Assembly

3.2. Assessing Fe/S Protein Function

3.2.1. Indirect Analysis of Lipoic Acid Synthase Activity

3.2.2. Assessing GPAT Autocatalytic Cleavage Activity

3.2.3. Probing for Alterations in Cellular Iron Homeostasis

4. Analysis of DPYD Enzyme Activity

4.1. Setup of the DPYD Enzymatic Reaction

4.1.1. Equipment.

4.1.2. Buffers and Reagents.

Notes:

Includes bibliographical references and indexes.

Description based on online resource; title from PDF title page (EBC, viewed February 19, 2018).

ISBN:

0-12-814718-0