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

1. Introduction

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

References

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

1. Introduction

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

Acknowledgments

References

Chapter Four: Iron-Sulfur Clusters in Zinc Finger Proteins

1. Introduction

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

Acknowledgments

References

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

1. Introduction

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

Acknowledgment

References

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

1. Introduction

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

Acknowledgments

References

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

1. Introduction

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

5. Summary and Conclusion

References

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

1. Introduction

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

2.1.1. Equipment

2.1.2. Buffers and Reagents

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

4.1.3. Procedure

4.1.4. Notes

4.2. Thin-Layer Chromatography and Autoradiography

4.2.1. Equipment

4.2.2. Buffers and Reagents

4.2.3. Procedure

4.2.4. Notes

5. Direct Determination of Fe/S Cluster Insertion Into Apoproteins by 55Fe Radiolabeling

5.1. 55Fe Coupling to Transferrin

5.1.1. Equipment

5.1.2. Buffers and Reagents

5.1.3. Procedure

5.1.4. Notes

5.2. 55Fe Radiolabeling of Tissue Culture Cells

5.2.1. Equipment

5.2.2. Buffers and Reagents

5.2.3. Procedure

5.2.4. Notes

5.3. Immunoprecipitation of Tagged and Radiolabeled 55Fe/S Proteins

5.3.1. Equipment

5.3.2. Buffers and Reagents

5.3.3. Procedure for Immunoprecipitation

5.3.4. Procedure for Reporter Protein Recovery

5.3.5. Notes

6. Summary and Conclusion

References

Chapter Nine: Steps Toward Understanding Mitochondrial Fe/S Cluster Biogenesis

1. Introduction

1.1. Mitochondrial Iron-Sulfur Clusters

2. Mitochondrial Iron-Sulfur Cluster Biogenesis

2.1. [2Fe—2S] Cluster Assembly by the ISU Complex

2.2. Transfer of [2Fe—2S] Clusters From the Isu1 Scaffold

2.3. [4Fe—4S] Cluster Formation by the ISA Complex

2.4. [4Fe—4S] Cluster Transfer

2.5. Unresolved Questions of Mitochondrial Fe/S Biogenesis

3. Defining New Components of Fe/S Cluster Biogenesis

3.1. Lessons From Past Fe/S Studies

4. Concluding Remarks

References

Chapter Ten: Approaches to Interrogate the Role of Nucleotide Hydrolysis by Metal Trafficking NTPases: The Nbp35-Cfd1 Iro ...

1. Introduction

1.1. Introduction to Cytosolic Iron-Sulfur Cluster Assembly and the Nbp35-Cfd1 Scaffold

1.2. The CIA Scaffold and Its Homologs Are Members of the Deviant Walker A Family of NTPases

2. Cloning, Expression, and Purification of the CIA Scaffold

2.1. Cloning Strategy

2.2. Protein Overproduction in E. coli

2.3. Purification of the CIA Scaffold

2.3.1. Equipment

2.3.2. Buffers and Reagents

2.3.3. Procedure for Purification of the Nbp35-Cfd1 Complex

2.4. Chemical Reconstitution of the FeS Clusters of the Nbp35-Cfd1 Heterodimer

2.4.1. Equipment

2.4.2. Buffers and Reagents

2.4.3. Procedure

2.5. Quantification of FeS Cluster Insertion

2.5.1. Equipment

2.5.2. Buffers and Reagents

2.5.3. Ferrozine Assay Procedure

2.5.4. Sulfide Assay Procedure

3. Kinetic Assays of Nucleotide Hydrolysis

3.1. Discontinuous Assay for Nucleotide Hydrolysis

3.2. Continuous Assay for Nucleotide Hydrolysis

3.2.1. Equipment

3.2.2. Buffers and Reagents

3.2.3. Coupled Enzyme Assay Procedure

4. Nucleotide Affinity Monitored via Fluorescence Spectroscopy

4.1. Nucleotide Affinity Evaluated via Fluorescence Anisotropy

4.1.1. Equipment

4.1.2. Buffers and Reagents

4.1.3. Procedure for FA mantATP Binding Experiment

4.2. Equilibrium Binding Assays With Trinitrophenyl-ATP

4.2.1. Equipment

4.2.2. Buffers and Reagents

4.2.3. Procedure for TNP-ATP Binding Assay

Acknowledgments

References

Chapter Eleven: Characterization of Glutaredoxin Fe-S Cluster-Binding Interactions Using Circular Dichroism Spectroscopy

1. Introduction

2. General Equipment

3. General Notes

4. Fe-S Cluster-Dependent Complex Formation

4.1. Characterization of Fe-S Cluster Binding in a Copurified Protein Complex

4.1.1. Buffers and Reagents

4.1.2. Procedure

4.1.3. Data Analysis

4.2. Characterization of Fe-S Cluster Complex Formation via Titration

4.2.1. Buffers and Reagents

4.2.2. Procedure

4.2.3. Data Analysis

4.2.4. Notes

5. Thermodynamic and Kinetic Characterization of Fe-S Cluster Transfer Reactions

5.1. CD-Monitored Thermodynamic Analysis of Fe-S Cluster Transfer Reactions

5.1.1. Additional Equipment

5.1.2. Buffers and Reagents

5.1.3. Procedure

5.1.4. Data Analysis

5.1.5. Notes

5.2. CD-Monitored Kinetic Analysis of Fe-S Cluster Transfer Reactions Based on Full Wavelength Scans

5.2.1. Additional Equipment

5.2.2. Buffers and Reagents

5.2.3. Procedure

5.2.4. Data Analysis

5.2.5. Notes

5.3. CD-Monitored Kinetic Analysis of Fe-S Cluster Transfer Reactions at a Single Wavelength

5.3.1. Additional Equipment

5.3.2. Procedure

5.3.3. Data Analysis

5.3.4. Notes

6. CD-Monitored pH Titrations of Fe-S Cluster Complexes

6.1. Additional Equipment

6.2. Buffers and Reagents

6.3. Procedure

6.4. Data Processing and pKa Determination

6.5. Notes

7. Summary and Conclusions

Acknowledgments

References

Chapter Twelve: Conformationally Gated Electron Transfer in Nitrogenase. Isolation, Purification, and Characterization of ...

1. Introduction

1.1. Biological and Abiological Nitrogen Fixation

1.2. Evidence for Gated-ET in Nitrogenase

1.3. Structural Dynamics of the FeP-MoFeP Complex

1.4. Redox-Dependent Structural Changes in the P-Cluster of A. vinelandii MoFe Protein

1.5. Redox-Dependent Structural Changes in the P-Cluster of G. diazotrophicus MoFe Protein

2. Materials and Methods

2.1. G. diazotrophicus as a Model Nitrogen-Fixing Organism

2.2. Equipment and Reagents

2.2.1. Major Equipment and Instruments Needed

2.2.2. Reagents Required for the Growth of G. diazotrophicus

2.2.3. Reagents Required for Nitrogenase Purification, Enzymatic Assays, and Crystallization

2.3. Growth Media for G. diazotrophicus Cultures

2.4. Freezer Stocks of G. diazotrophicus

2.5. Nitrogenase Expression in G. diazotrophicus

2.5.1. General Growth Considerations

2.5.2. Factors Affecting Nitrogenase Expression in G. diazotrophicus

2.5.3. Monitoring Nitrogenase Activity in Whole Cells

2.6. Buffers and Solutions for G. diazotrophicus Lysis and Nitrogenase Purification

2.7. Isolation and Purification of G. diazotrophicus Nitrogenase

2.7.1. General Procedures for Working With Nitrogenase

2.7.2. Lysis

2.7.3. Purification

2.8. Enzymatic Activity Assays for Gd-Nitrogenase

2.9. Electron Paramagnetic Resonance Spectroscopy of G. diazotrophicus MoFe Protein

2.10. Crystallography of G. diazotrophicus MoFe Protein

2.10.1. Crystallization

2.10.2. Data Collection, Data Processing, and Structural Refinement

2.10.3. Structural Refinement

3. Conclusion

Acknowledgments

References

Chapter Thirteen: Protein Film Electrochemistry of Iron-Sulfur Enzymes

1. Introduction

2. Technical Aspects

3. Examples in Which Specific FeS Clusters in Proteins Are Studied

4. Steady-State Catalytic Electron Flow Through FeS Cluster Relays

5. A Case Study in Control: The Final Stages of H-Cluster Assembly in Hydrogenases

6. Summary

Acknowledgments

References

Chapter Fourteen: NRVS for Fe in Biology: Experiment and Basic Interpretation

1. Introduction

2. Materials

2.1. Calibration

3. Procedure

4. Application

4.1. Rubredoxin

4.2. [FeFe] Hydrogenase

5. Perspective

References

Chapter Fifteen: Advanced X-ray Spectroscopic Methods for Studying Iron-Sulfur-Containing Proteins and Model Complexes

1. Introduction

2. X-ray Absorption Spectroscopy

2.1. Metal K-Edge XAS

2.2. Ligand K-Edge XAS

2.3. Metal and Ligand K-Edge XAS Experimental Considerations

3. X-ray Emission Spectroscopy

3.1. XES

3.2. Kβ XES

3.3. Valence-to-Core XES

3.4. XES Experimental Considerations

4. RXES/RIXS Measurements in the Hard X-ray Regime

4.1. 1s2p RIXS

4.2. 1s3p RIXS

4.3. 1s-VtC RIXS

4.4. Experimental Considerations

5. RIXS and XMCD Measurements in the Soft X-ray Regime

6. Summary and Outlook

Acknowledgments

References

Author Index

Subject Index

Back Cover