Oxygen Sensing: Methods and Protocols

Preface
Contents
Contributors
Chapter 1: Integrating UV-Vis Spectroscopy and Oxygen Optode for Accurate Determination of Oxygen Affinity of Proteins
1 Introduction
2 Materials
2.1 UV-Vis Spectroscopy and Integrated Oxygen-Optode Sensor
2.2 Oxygen Calibration with Chlorite/Chlorite Dismutase (Cld)
2.3 Measuring the Oxygen Affinity of Cs H-NOX
3 Methods
3.1 Assembling the Spectroscope and Oxygen Optode Sensor
3.2 Calibration of Oxygen Optode Sensor with Chlorite/Chlorite Dismutase
3.3 Measuring the Oxygen Affinity of Cs H-NOX
4 Notes
References
Chapter 2: Measurement of O2 Binding by Sensory Hemeproteins
1 Introduction
2 Materials
2.1 Proteins
2.2 Solutions and Reagents
2.3 Equipment
3 Methods
3.1 Pyridine Hemochromogen Assay of Heme Content
3.2 Basis Spectra
3.2.1 Deoxy-State, FeII
3.2.2 Oxy-State, FeIIO2
3.2.3 Met-State, FeIII
3.3 Autoxidation Rate Constant, kox
3.4 Equilibrium Dissociation Constant, Kd, for Binding of O2
3.4.1 Direct Titration
3.5 Binding by Competition
3.5.1 Obtain an Apparent AppKd
3.6 Dissociation Rate Constant, koff, for Binding of O2
3.7 Association Rate Constant, kon, for Binding of O2
3.7.1 Measurement by Flash Photolysis
3.7.2 Measurement by Stopped Flow
3.7.3 Data Analysis
4 Conclusions and General Considerations
5 Notes
References
Chapter 3: Resonance Raman Characterization of O2-Binding Heme Proteins
1 Introduction
2 Materials
2.1 Sample Preparation
2.2 Resonance Raman Setup
3 Methods
3.1 Preparing Oxy Adducts of Protein Samples
3.2 Resonance Raman Measurements
4 Notes
References
Chapter 4: Anaerobic Infrared Spectroelectrochemical Methods for Studying Oxygen-Sensitive [FeFe] Hydrogenases
1 Introduction
2 Materials
2.1 Fabrication of the Spectroelectrochemical Cell
2.2 Protein Expression and Isolation
2.3 Protein Activation
2.4 Infrared Spectrometry and Electrochemistry
3 Methods
3.1 IR Cell Assembly
3.2 IR Spectroelectrochemical Measurements
3.3 Data Processing
3.4 Data Analysis
4 Additional Remarks
References
Chapter 5: Monitoring the Kinase Activity of Heme-Based Oxygen Sensors and Its Dependence on O2 and Other Ligands Using Phos-T...
1 Introduction
2 Materials
2.1 Solutions of Heme Proteins and Other Related Reagents
2.2 Sodium Dodecyl Sulfate (SDS) Polyacrylamide Gel Containing Phos-tag
3 Methods
3.1 Incubations Containing the Various Heme Iron Oxidation and Ligand States Influencing the AfGcHK Autophosphorylation and RR...
3.2 10% SDS-PAGE in the Presence of 75 μM Phos-Tag Acrylamide and 0.2 mM MnCl2
4 Notes
References
Chapter 6: In Vitro Measurement of Gas-Dependent and Redox-Sensitive Diguanylate Cyclase Activity
1 Introduction
2 Materials
2.1 Reagents and Equipment
2.2 Pyrophosphate Assay Kit Preparation
3 Methods
3.1 Pyrophosphate Assay Protein Preparation
3.2 Master Mix Preparation
3.3 Reaction Initiation
3.4 Spectrophotometric Analysis and Michaelis-Menten Kinetics
3.5 Challenges
4 Notes
References
Chapter 7: Spectrophotometric Method for the Quantification and Kinetic Evaluation of In Vitro c-di-GMP Hydrolysis in the Pres...
1 Introduction
2 Materials and Equipment
2.1 EnzChek Phosphate Detection Kit
2.2 Four Concentrations of Purified PDE-Containing Protein at 3x the Desired Final Concentration (μM) (See Note 2)
2.3 Absorbance Plate Readers
3 Methods
3.1 PDE Enzyme Activity Master Mix and Pi Standard Reaction Mix and Plate Assembly
3.2 Reaction Initiation and Spectrophotometric Monitoring
3.3 Analysis of Spectrophotometric Data to Generate Michaelis-Menten Data (Vmax, KM, and kcat)
3.3.1 Generate Standard Curve
3.4 Generate Initial Rate Plots
3.5 Determine vmax of PDE-Containing Enzyme
3.6 Determine the kcat of the PDE-Containing Enzyme
4 Notes
References
Chapter 8: Hydrogen/Deuterium Exchange Mass Spectrometry of Heme-Based Oxygen Sensor Proteins
1 Introduction
2 Materials
2.1 Heme Redox and Ligation State Modification
2.2 Hydrogen/Deuterium Exchange: Sample Collection
2.3 LC-MS (/MS) Analysis and Data Interpretation
3 Methods
3.1 Optimization of the Digestion Conditions
3.2 Sample Preparation and LC-MS/MS Analysis
3.3 Hydrogen/Deuterium Exchange
3.4 LC-MS Analysis
3.5 HDX-MS Data Interpretation, Visualization, and Reporting
4 Notes
References
Chapter 9: Methods for Biophysical Characterization of SznF, a Member of the Heme-Oxygenase-Like Diiron Oxidase/Oxygenase Supe...
1 Introduction
2 Materials
2.1 Overexpression of SznF
2.2 Purification of SznF
2.2.1 Ni-NTA Affinity Chromatography
2.2.2 Size-Exclusion Chromatography
2.3 Metal Analysis of SznF by ICP-AES
2.4 Deoxygenating and Oxygenating Solutions
2.5 Stopped-Flow Absorption Spectroscopy
2.6 Mössbauer Spectroscopy
2.7 X-Ray Crystallographic Characterization of SznF
3 Methods
3.1 Overexpression of SznF in M9 Medium with Mn(II) Supplementation
3.1.1 Preparation of a Seed Culture
3.1.2 Preparation of a Starter Culture
3.1.3 Large-Scale Overexpression
3.1.4 Incorporation of L-SeMet
3.2 Purification of SznF
3.2.1 Ni-NTA Affinity Chromatography
3.2.2 Size-Exclusion Chromatography
Degassing Solutions for FPLC
Preparing Pure SznF for X-Ray Crystallography
3.3 Metal Analysis of SznF by ICP-AES
3.4 Characterization of the SznF-Peroxo-Fe(III)2 Complex
3.4.1 Stopped-Flow Absorption (SF-Abs) Spectroscopy
Deoxygenating Buffers
Deoxygenating Protein Solutions
Oxygenating Buffer
Detection of a Transient Intermediate in SznF
3.4.2 Mössbauer Spectroscopy
Setting Up the Freeze-Quench Bath
Preparation of 57Fe Stock
Rapid Freeze-Quench Sample Preparation
3.4.3 X-Ray Crystallographic Characterization of SznF
Argon Exchanging Solutions
Obtaining an Apo Structure of SznF
Obtaining a Holo Structure of SznF
Harvesting Crystals
Data Collection
4 Notes
References
Chapter 10: Analyzing Iron and Oxygen-Regulated Protein Complex Formation Using Proteomic Mass Spectrometry
1 Introduction
2 Materials
3 Methods
3.1 Treatment of Mammalian Cell Lines with Low Oxygen Tension or a Chemical Inducer of Hypoxia
3.1.1 Seeding Cells in Sterile Cell Culture Dishes for Hypoxia Treatment
3.1.2 Preparing Cells Prior to Hypoxia Treatment
3.2 Assembly of the Hypoxia Chamber
3.3 Purging the Hypoxia Chamber with the Premixed Gas Mixture
3.3.1 Maintaining Control Cells for Normoxia Treatment
3.4 Post-Hypoxia Processing of Cells
3.5 Induction of Hypoxia by Treatment of Cells with Chemical Inducer of Hypoxia
3.6 Proteomic Characterization of the Interactome of the Iron-Sensing Protein BXL5 in Response to Hypoxia Treatment
3.6.1 Preparation of Cells and Affinity Purification of Tagged FBXL5 for APMS
3.6.2 Precipitation of Protein from Eluates Using Trichloroacetic Acid
3.6.3 Reduction, Alkylation, and Digestion of Precipitated Proteins
3.6.4 Desalting of Digested Peptides Prior to Mass Spectrometry Analysis
4 Note
References
Chapter 11: CRISPR Activator Approaches to Study Endogenous Androglobin Gene Regulation
1 Introduction
2 Materials
2.1 Cloning
2.2 Transfection of Mammalian Cells
2.3 RNA Extraction and cDNA Generation
2.4 Real-Time Quantitative PCR (qPCR)
3 Methods
3.1 In Silico Analysis of Regulatory Elements
3.2 Plasmid Construction
3.2.1 Cloning of sgRNAs Targeting Genomic Region of Interest
3.2.2 Cloning of Transcription Factors
3.3 Plasmid Delivery into Mammalian Cell Lines
3.3.1 Transfection of HEK293T, MCF-7, and HeLa Cells with ROTI-Fect
3.3.2 Calcium Phosphate Method of Transfection in HEK293T Cells
3.4 Cell Lysis and Sample Preparation
3.4.1 RNA Extraction
3.4.2 Reverse Transcription
3.5 Real-Time PCR (qPCR)
4 Notes
References
Chapter 12: Assays to Study Hypoxia-Inducible Factor Prolyl Hydroxylase Domain 2 (PHD2), a Key Human Oxygen Sensing Protein
1 Introduction
2 Materials
2.1 Reagents and Solutions for Recombinant PHD2 Production and Purification
2.1.1 Cloning and Expression Vector Construction
2.1.2 Production of Recombinant PHD2181-426
2.1.3 Purification of Recombinant PHD2181-426
Purification by Immobilized Metal Affinity Chromatography (IMAC)
Apo-Protein Preparation
Purification by Size-Exclusion Chromatography
2.2 Reagents and Solutions to Study PHD2 Activity
2.3 Reagents and Solutions for HIF Immunoblotting
2.3.1 Urea-SDS Lysis Buffer (See Note 3)
2.3.2 SDS Polyacrylamide Gel
2.3.3 Other Buffers, Solutions and Consumables
3 Methods
3.1 Recombinant PHD2 Production and Purification
3.1.1 Cloning of Recombinant Plasmid Encoding PHD2181-426
Preparation of Gene Fragment Insert to Generate Cohesive Ends
Preparation of Vector (Cutting and Cohesive End Generation)
Annealing and Transformation
3.1.2 Production and Purification of Recombinant PHD2181-426
Production of Recombinant PHD2181-426
Initial Purification by IMAC
Apo-Protein Preparation
Final Purification by Size-Exclusion Chromatography
3.2 PHD2 Activity Assay
3.2.1 Setting Up the PHD2 Reaction
3.2.2 Monitoring Peptide Hydroxylation by MALDI-MS
3.2.3 Sample Preparation for MALDI-TOF Measurements
3.3 HIF Immunoblotting as a Measure of PHD Activity in Cells
3.3.1 Cell Seeding and Treatment
3.3.2 Cell Harvesting
3.3.3 Preparation of SDS Polyacrylamide Gels
3.3.4 SDS Polyacrylamide Gel Electrophoresis
3.3.5 Electrophoretic Transfer
3.3.6 Total Protein Staining with Ponceau S
3.3.7 Blocking and Antibody Incubation
4 Notes
References
Chapter 13: Kinetic Measurements to Investigate the Oxygen-Sensing Properties of Plant Cysteine Oxidases
1 Introduction
2 Materials
2.1 Medium and Supplements for Protein Expression
2.2 Buffers, SDS-PAGE, and Materials Used During Protein Purification
2.3 Buffers, Stocks, and Equipment Used for the Initial Activity Assay
2.4 Buffers and Equipment for O2-Dependent Kinetic Assays
3 Methods
3.1 Expression of Recombinant AtPCO4 in Bacterial Cells
3.2 Purification of AtPCO4
3.3 Initial Activity Assay
3.4 Optimization of Enzyme Concentration
3.5 Optimization of Iron and Ascorbate Concentrations
3.5.1 Optimization of the Iron Concentration
3.5.2 Optimization of the Ascorbate Concentration
3.6 Steady-State Kinetic Assays with Substrate Peptide
3.7 O2-Dependent Kinetic Assays
3.7.1 Setup of the O2-Exposure Experiment
3.7.2 Steady-State Kinetic Assays with O2
4 Notes
References
Chapter 14: Methods for Culturing Anaerobic Microorganisms
1 Introduction
2 Materials
2.1 Culture Media Components
3 Methods
3.1 Preparation of Culture Media
3.2 Pre-inoculation
3.3 Inoculation and Growth
4 Notes
References
Index