Note: Cover -- Two-Component Signaling Systems, Part B -- Copyright -- Table of Contents -- Contributors to Volume 423 -- Methods in Enzymology -- Section I Structural Approaches -- [1]. The PICM Chemical Scanning Method for Identifying Domain-Domain and Protein-Protein Interfaces: Applications to the Core Signaling Complex of E. coli Chemotaxis -- Abstract -- Introduction -- Comparison of the PICM Method with Other Scanning Approaches -- PICM Studies of the Core Signaling Complex of Bacterial Chemotaxis -- Generalizing the PICM Method to Map Docking Sites in Other Systems -- Incorporation of an Affinity Tag and Creation of a Cysless Protein -- Choice of Positions for Cys Incorporation and Creation of a Mutant Library -- Selection of a Cys-Specific Probe for Chemical Modification -- Probe Labeling and Purification of the Single Cys Mutants -- Quantitation of Probe Coupling -- Measuring Functional Effects of Cys Substitution and Bulky Probe Coupling -- Interpretation of Results-Mapping Out Docking Sites -- Acknowledgments -- References -- [2]. Use of Site-Directed Cysteine and Disulfide Chemistry to Probe Protein Structure and Dynamics: Applications to Soluble and Transmembrane Receptors of Bacterial Chemotaxis -- Abstract -- Introduction -- Site-Directed Cysteine and Disulfide Chemistry: History -- Site-Directed Cysteine and Disulfide Chemistry: Applications and Limitations -- Incorporation of an Affinity Tag and Creation of a Cysless Protein -- Choice of Positions for Cys Incorporation and Creation of a Mutant Library -- Analysis of 2˚ deg Structure by Chemical Reactivity Scanning -- Disulfide Mapping of Spatial Proximity and Conformational Changes -- Disulfide Trapping of Thermal Backbone and Domain Motions -- Acknowledgments -- References -- [3]. Measuring Distances by Pulsed Dipolar ESR Spectroscopy: Spin-Labeled Histidine Kinases -- Abstract. , Introduction -- Dipolar ESR Spectroscopy -- Case Study: PDS Reconstruction of Histidine Kinases Signaling Complex -- Concluding Remarks -- Acknowledgments -- References -- [4]. Rigid Body Refinement of Protein Complexes with Long-Range Distance Restraints from Pulsed Dipolar ESR -- Abstract -- Introduction -- Method -- Initial Conformation of the Complex -- Results -- Discussion -- Acknowledgments -- References -- [5]. TonB/TolA Amino-Terminal Domain Modeling -- Abstract -- Introduction -- Alanyl Replacement -- TonB/TolA Chimeras -- Acknowledgments -- References -- [6]. Functional Dynamics of Response Regulators Using NMR Relaxation Techniques -- Abstract -- Introduction -- The Experimental Setup -- Two-State Allosteric Activation Identified by NMR Chemical Shift Analysis -- Two-State Allosteric Activation Buttressed by Standard NMR Relaxation Experiments -- A New Approach for Quantitative Analysis of Microsecond Protein Dynamics -- Conclusions -- Acknowledgments -- References -- [7]. The Design and Development of Tar-EnvZ Chimeric Receptors -- Abstract -- Introduction -- Construction of Tar-EnvZ Chimeric Protein, Taz -- Asp-Dependent Induction of ompC-lacZ Fusion Gene by Taz and OmpR -- Phenotype Analysis of the Taz Construct -- Regulation of Binding of Asp to One of Two Asp-Binding Pockets of Tar Receptor to Study Signal Transduction -- The Right Configuration of HAMP Domain is Crucial for Proper Signal Transduction in a Tar-EnvZ Chimeric Protein -- Conclusions -- Acknowledgments -- References -- [8]. Functional and Structural Characterization of EnvZ, an Osmosensing Histidine Kinase of E. coli -- Abstract -- Introduction -- Expression and Purification of EnvZc -- Expression and Purification of Domain A and Domain B -- Characterization of EnvZc -- Characterization of EnvZ with Help of its Specific Mutants. , Creation of a Monomeric Histidine Kinase Using EnvZc -- NMR Structural Analysis of Domain A and Domain B -- Conclusion -- Acknowledgments -- References -- [9]. Light Modulation of Histidine-Kinase Activity in Bacterial Phytochromes Monitored by Size Exclusion Chromatography, Crosslinking, and Limited Proteolysis -- Abstract -- Introduction -- Sample Preparation -- Photoconversion, Experimental Light Conditions, Protein Concentration -- Size Exclusion Chromatography -- Protein Crosslinking -- Limited Proteolysis -- Autophosphorylation -- References -- [10]. A Temperature-Sensing Histidine Kinase-Function, Genetics, and Membrane Topology -- Abstract -- Introduction -- Genetic Approaches to Characterize CorRSP -- Transcriptional Analysis -- Biochemical Characterization of CorRSP -- Topological Analysis of the HPK CorS -- Concluding Remarks -- Acknowledgments -- References -- [11]. The Regulation of Histidine Sensor Kinase Complexes by Quorum Sensing Signal Molecules -- Abstract -- Introduction -- Bacterial Quorum Sensing -- The V. harveyi AI-2 Signal Transduction Pathway -- Regulation of the LuxPQ Receptor Complex by AI-2 -- Expression of Wild-Type and Mutant LuxPQp -- Purification of LuxP, LuxQp, and LuxPQp -- Crystallization of LuxPQp Complexes -- Functional Analysis -- Conclusions -- Acknowledgment -- References -- Section II. Reconstitution of Heterogeneous Systems -- [12]. Liposome-Mediated Assembly of Receptor Signaling Complexes -- Abstract -- Introduction -- Results―Biochemical Activity of Liposome-Assembled Receptor Fragments -- Methods -- Conclusion -- Acknowledgment -- References -- [13]. Analyzing Transmembrane Chemoreceptors Using In Vivo Disulfide Formation Between Introduced Cysteines -- Abstract -- Introduction -- Disulfide Formation In Vivo: Applications and Limitations -- Oxidation Reagents. , Oxidation Treatments That Preserve In Vivo Function -- Experimental Designs -- Procedures -- Analysis -- Closing Comments -- Acknowledgments -- References -- [14]. Using Nanodiscs to Create Water-Soluble Transmembrane Chemoreceptors Inserted in Lipid Bilayers -- Abstract -- Introduction -- Developing a Protocol for Producing Nanodisc-Embedded Protein -- Preparation of Nanodisc-Embedded Chemoreceptor -- Preparation of Cytoplasmic Membranes with High Tar-6H Content -- Receptor Purification -- Preparation of Receptor-Containing Nanodiscs -- Analysis of Receptor-Containing Nanodiscs -- Acknowledgments -- References -- [15]. Assays for CheC, FliY, and CheX as Representatives of Response Regulator Phosphatases -- Abstract -- Introduction -- Assays -- Phosphate Release Assay -- Pulldowns -- Acknowledgments -- References -- [16]. Genetic Dissection of Signaling Through the Rcs Phosphorelay -- Abstract -- Overview -- Flowchart of Testing: Signaling Inputs -- Analysis of the Regulation of a Target Gene -- Analysis of Signaling via the Rcs Phosphorelay -- RcsC-Dependent Signaling -- RcsA-Dependent Signaling: Increased RcsA Synthesis or Stability -- Determining Whether a Strain Carries a lon Mutation or is Phenotypically Lon¯ -- Conclusions -- Acknowledgments -- References -- Section III Intracellular Methods and Assays -- [17]. In Vivo Measurement by FRET of Pathway Activity in Bacterial Chemotaxis -- Abstract -- Introduction -- FRET -- FRET Measurement of the Interaction Between CheY-YFP and CheZ-CFP in a Population of Bacteria Fixed to a Microscope Cover Slip -- FRET Measurement of the Interaction Between CheY-YFP and CheZ-CFP in Single Bacteria Fixed to a Microscope Cover Slip -- BRET Measurement of the Interaction Between YFP-CheY and -CheZ-RLUC in a Population of Bacteria Swimming in a Cuvette. , Comparison of Different Approaches and Application to Other Two-Component Systems -- References -- [18] In Vivo and In Vitro Analysis of the Rhodobacter sphaeroides Chemotaxis Signaling Complexes -- Abstract -- Introduction -- In Vitro Analysis of Signaling by the Kinase Cluster -- Genomic Replacements with Fluorescent Protein Fusions for Studying Protein Localization -- Assessing the Functionality of the Fluorescent Protein Fusions -- Summary -- References -- [19] In Vivo Crosslinking Methods for Analyzing the Assembly and Architecture of Chemoreceptor Arrays -- Abstract -- Introduction -- Use of a Lysine-Targeted Crosslinker to Probe Receptor-Receptor Interactions in Cells -- Use of Cys-Targeted Crosslinking to Probe for the Trimer-of -Dimers Geometry in Cellular Chemoreceptor Assemblies -- Intracytoplasmic Disulfide Crosslinks -- A Trifunctional Cys-Targeted Crosslinker -- TMEA Competition Assay: A Tool for Assessing the Trimer-Forming Ability of Mutant Receptors -- Exchange Assay: Dynamic Changes in Trimer Composition as a Consequence of Changes in the Receptor Population -- Concluding Remarks -- Acknowledgments -- References -- [20]. A "Bucket of Light" for Viewing Bacterial Colonies in Soft Agar -- Abstract -- Viewing Colonies Grown in Soft Agar -- Building a Bucket of Light -- Acknowledgments -- References -- [21]. Phenotypic Suppression Methods for Analyzing Intra- and Inter-Molecular Signaling Interactions of Chemoreceptors -- Abstract -- Introduction -- Conformational Suppression within Receptor Molecules -- Conformational Suppression Between Receptor Molecules -- Acknowledgments -- References -- [22]. Single-Cell Analysis of Gene Expression by Fluorescence Microscopy -- Abstract -- Introduction -- Transcriptional Reporters -- Measuring Cellular Fluorescence by Microscopy -- Agarose Pads -- Fluorescence Microscopy and Image Acquisition. , Image Analysis.

Note: Front Cover -- Two-Component Signaling Systems, Part A -- Copyright Page -- Table of Contents -- Contributors to Volume 422 -- Volumes in Series -- Section I: Computational Analyses of Sequences and Sequence Alignments -- Chapter 1: Comparative Genomic and Protein Sequence Analyses of a Complex System Controlling Bacterial Chemotaxis -- Introduction -- Bioinformatics Tools and Resources for Identifying and Analyzing Chemotaxis Components -- Defining MCP Membrane Topology -- Diversity of Input (Sensory) Domains in MCPs -- HAMP Domain Identification -- MCP Signaling Domain -- MCP Pentapeptide Tether -- The CheA Histidine Kinase: Domain Organization, Conservation, and Diversity -- The CheY Response Regulator: Big Problems of the Small Protein -- CheB and CheR -- CheC and CheX -- CheD -- CheZ -- CheW and CheV -- References -- Chapter 2: Two-Component Systems in Microbial Communities: Approaches and Resources for Generating and Analyzing Metagenomic Data Sets -- Introduction -- Generating Metagenomic Data -- Assembly of Environmental Sequence Data -- Gene Prediction in Environmental Sequence Data -- Analysis of Two-Component System Genes in Environmental Sequence Data -- Acknowledgments -- References -- Chapter 3: Identification of Sensory and Signal-Transducing Domains in Two-Component Signaling Systems -- Introduction -- Computational Tools for Domain Identification -- Sequence Analysis of Histidine Kinases -- Sequence Analysis of Response Regulators -- Sequence Analysis of Prokaryotic Signal Transducers -- Functional Annotation of Multidomain Proteins -- References -- Chapter 4: Features of Protein-Protein Interactions in Two-Component Signaling Deduced from Genomic Libraries -- Introduction -- Identifying Coupled Columns -- Predicting Protein-Protein Interaction -- Summary -- Acknowledgments -- References. , Chapter 5: Sporulation Phosphorelay Proteins and Their Complexes: Crystallographic Characterization -- Introduction -- Methods -- Insights from Structural Analysis -- Conclusion -- Acknowledgments -- References -- Chapter 6: Control Analysis of Bacterial Chemotaxis Signaling -- Introduction -- Basic Concepts in Dynamics and Mathematical Modeling -- Robustness and Steady-State Sensitivity Analysis -- Constructing and Interpreting a Bode Plot -- Primer on Integral Feedback Control -- Noise Filtering and the Kalman Filter -- Future Perspectives and Further Information -- References -- Chapter 7: Classification of Response Regulators Based on Their Surface Properties -- Introduction -- Classification of the Receiver Domain of RRs Using Protein Interaction Surfaces -- Modeling and Subclassification of Receiver Domains of OmpR Subfamily RRs in B. subtilis and E. coli -- Modeling and Subclassification of the Receiver Domain of RRs in V. vulnificus -- References -- Section II: Biochemical and Genetic Assays of Individual Components of Signaling Systems -- Chapter 8: Purification and Assays of Rhodobacter capsulatus RegB-RegA Two-Component Signal Transduction System -- Introduction -- Expression and Purification of RegB -- Expression and Purification of RegA -- RegB Kinase and Phosphotransfer Assays -- Acknowledgment -- References -- Chapter 9: Purification and Reconstitution of PYP-Phytochrome with Biliverdin and 4-Hydroxycinnamic Acid -- Introduction -- Vector Construct -- Prepation of 4-Hydroxycinnamic Acid Anhydride and Biliverdin -- Overexpression and Reconstitution of apo-Ppr with Chromophores -- Purification of Ppr Reconstituted with Chromophores -- Spectroscopic Measurements of holo-Ppr, Ppr-BV, and Ppr-pCA -- In Vitro Autophosphorylation of Ppr -- Acknowledgments -- References. , Chapter 10: Oxygen and Redox Sensing by Two-Component Systems That Regulate Behavioral Responses: Behavioral Assays and Structural Studies of Aer Using In Vivo Disulfide Cross-Linking -- Introduction -- Assays of Oxygen and Redox Sensing: General Considerations -- Temporal Assay for Aerotaxis -- Spatial-Gradient Capillary Assay for Aerotaxis -- Using a Capillary to Determine the Preferred Partial Pressure of Oxygen for Bacteria -- Spatial-Gradient Soft Agar Plate Assays for Aerotaxis -- Spatial Assays for Redox Taxis -- Temporal Assay for Redox Taxis -- Disulfide Cross-Linking In Vivo to Elucidate the Structure of Aer -- Site-Directed Mutagenesis for Cysteine Replacement -- Disulfide Cross-Linking in the Cytosol Using Copper Phenanthroline -- Differentiating Intra- from Interdimeric Disulfide Bonds -- In Vivo Cross-Linking Using Bifunctional Sulfhydryl-Reactive Linkers -- Determining the Boundaries of Transmembrane Segments in Receptors -- Accessibility Studies in Membrane Vesicles -- Acknowledgments -- References -- Chapter 11: Two-Component Signaling in the Virulence of Staphylococcus aureus: A Silkworm Larvae-Pathogenic Agent -- Introduction -- Silkworm Larvae Infection Model -- Pathogenicity-Related Genes That Can Be Identified in the Silkworm Infection Assay -- Identification of Genes Involved in the Killing of Silkworm Larvae by Bacteria -- Identification of an SA0614 Response Regulator Mutant by Monitoring CPZ Sensitivity and Ability to Kill Silkworm Larvae -- Silkworm Larvae Infection Assay -- Measurement of the Number of Bacteria in Silkworm Hemolymph -- Defect in Cell Wall Integrity of the SA0614 Response Regulator Mutant -- Detergent and Lysozyme Sensitivity Test -- Melanization-Inducing Activity of Bacterial Peptidoglycan -- References -- Chapter 12: TonB System, In Vivo Assays and Characterization -- Introduction. , Selection For and Against the tonB Gene -- Precautions for Experiments Where TonB System Proteins Are Expressed from Plasmids -- Phenotypic Assays for the TonB System -- Mechanistically Informative Assays -- Potentially Mechanistically Informative Assays -- Acknowledgments -- References -- Chapter 13: Biochemical Characterization of Plant Ethylene Receptors Following Transgenic Expression in Yeast -- Introduction -- Transgenic Expression of Ethylene Receptors in Yeast -- Histidine Kinase Activity -- Isolation of Receptors for Use in Ethylene-Binding Assays -- Ethylene-Binding Activity -- Considerations When Working with Mercuric Perchlorate -- Acknowledgments -- References -- Chapter 14: Structure of SixA, a Histidine Protein Phosphatase of the ArcB Histidine-Containing Phosphotransfer Domain in Escherichia Coli -- Introduction -- Overall Structure -- Conservation of Catalytic Machinery and Active Site -- Sequence Analysis of SixA Homologs -- Eukaryotic Histidine Phosphatases -- Acknowledgments -- References -- Chapter 15: Triggering and Monitoring Light-Sensing Reactions in Protein Crystals -- Light-Regulated Histidine Kinases: A Brief Introduction -- Microbial Rhodopsins -- PAS/GAF/LOV Domains -- Photoreceptors and Kinetic Crystallography: A Near Perfect Match -- Kinetic Crystallography: Two Alternative Strategies -- Trapping Intermediates for X-Ray Crystallography -- Structural Interpretation of Kinetic Crystallography Results -- Optical Properties of Protein Crystals -- Mounting Crystals -- Design of a Single Crystal Microspectrophotometer -- Challenges of Recording UV-Visible Absorption Spectra in Crystals -- Leaking Light Introduces Spectral and Kinetic Artifacts -- Fluorescence Measurements -- Microspectrophotometry: Summary and Warning -- Light Activation of Photoreceptor Crystals -- Aligning the Activating Light Beam and the Crystal Position. , Summary and Outlook -- References -- Chapter 16: Synthesis of a Stable Analog of the Phosphorylated Form of CheY: Phosphono-CheY -- Introduction -- Protocols -- Acknowledgments -- References -- Chapter 17: Application of Fluorescence Resonance Energy Transfer to Examine EnvZ/OmpR Interactions -- Introduction -- Overexpression of EnvZ and Preparation of Spheroplasts -- Protein Purification and Fluorescent Labeling of OmpR -- Fluorescence Resonance Energy Transfer -- OmpR Has a Higher Affinity for EnvZ Than OmpR~P -- Concluding Remarks -- Acknowledgments -- References -- Chapter 18: Gene Promoter Scan Methodology for Identifying and Classifying Coregulated Promoters -- Introduction -- Challenge of Identifying Promoter Features Governing Gene Transcription -- GPS Methodology as an Integrated Algorithm -- Exploring Targets of Regulation of a Response Regulator Using GPS -- Technical Specifications of GPS -- Uncovering Promoter Profiles Regulated by Response Regulator PhoP Using GPS -- Conclusions -- Acknowledgments -- References -- Chapter 19: Targeting Two-Component Signal Transduction: A Novel Drug Discovery System -- Introduction -- Differential Growth Assay -- High-Throughput Genetic System -- Acknowledgments -- References -- Chapter 20: The Essential YycFG Two-Component System of Bacillus subtilis -- Introduction -- Construction of Conditional Mutants -- Transposon Mutagenesis to Identify Regulatory Elements -- Constructing In-Frame Deletions in the yyc Operon -- Studying Interactions between the YycG Kinase and Its Regulatory Proteins -- Subcellular Localization Studies -- Concluding Remarks -- Acknowledgments -- References -- Section III: Physiological Assays and Readouts. , Chapter 21: Isolation and Characterization of Chemotaxis Mutants of the Lyme Disease Spirochete Borrelia burgdorferi Using Allelic Exchange Mutagenesis, Flow Cytometry, and Cell Tracking.

Note: Front Cover -- Methods in Enzymology Two-Component Signaling Systems, Part C -- Copyright Page -- Contents -- Contributors -- Chapter 1: Characterizing Cross-Talk In Vivo: Avoiding Pitfalls and Overinterpretation -- Abstract -- 1. Overview -- 2. Sources of Cross-Talk -- 3. Cross-Talk Suppression -- 4. Transcriptional Reporters -- 5. Response Regulator Localization -- 6. Phosphatase Cross-Talk -- 7. Signal Response in Cross-Talk Networks -- 8. Concluding Remarks -- Acknowledgments -- References -- Chapter 2: Inference of Direct Residue Contacts in Two-Component Signaling -- Abstract -- 1. Introduction -- 2. Extraction Tools -- 3. DCA: Direct Coupling Analysis -- Appendix: Nonstandard Linear Algebra Functions -- Acknowledgments -- References -- Chapter 3: Computational Modeling of Phosphotransfer Complexes in Two-Component Signaling -- Abstract -- 1. Introduction -- 2. Methods -- 3. Summary -- Acknowledgments -- References -- Chapter 4: Kinetic Studies of the Yeast His-Asp Phosphorelay Signaling Pathway -- Abstract -- 1. Introduction -- 2. Materials and Methods -- 3. Conclusion -- Acknowledgments -- References -- Chapter 5: Purification of MBP-EnvZ Fusion Proteins Using an Automated System -- Abstract -- 1. Introduction -- 2. Comparison Between the Cytoplasmic Domains of E. coli and Typhi EnvZ Proteins -- 3. Purification of the Recombinant MBP Proteins by FPLC -- 4. SDS-PAGE Analysis of Recombinant Expressed MBP-EnvZc Protein -- 5. Results -- 6. Discussion -- Acknowledgments -- References -- Chapter 6: Measurement of Response Regulator Autodephosphorylation Rates Spanning Six Orders of Magnitude -- Abstract -- 1. Overview of Response Regulator Autodephosphorylation -- 2. Purification of Response Regulator Proteins -- 3. General Considerations for Autodephosphorylation Assays -- 4. Assay of Autodephosphorylation by Loss of 32P. , 5. Assay of Autodephosphorylation by Fluorescence -- 6. Assay of Autodephosphorylation by Pi Release -- 7. Assay of Autodephosphorylation from Systems of Reactions -- 8. Future Prospects -- Acknowledgments -- References -- Chapter 7: Transmembrane Receptor Chimeras to Probe HAMP Domain Function -- Abstract -- 1. Introduction -- 2. Design of Chimeras of HAMP Domains Fused to the AC Rv3645 Catalytic Domain -- 3. Choice of Vector and Cloning Strategy -- 4. Expression and Purification of the Chimeras -- 5. AC Assay -- 6. Example of Applications of the Method and Results -- 7. Concluding Remarks -- Acknowledgments -- References -- Chapter 8: Light-Activated Bacterial LOV-Domain Histidine Kinases -- Abstract -- 1. Introduction -- 2. Description of Method -- 3. Concluding Remarks and Future Perspectives -- Acknowledgments -- References -- Chapter 9: Characterization of Bacteriophytochromes from Photosynthetic Bacteria -- Abstract -- 1. Introduction -- 2. Cloning BphP in Expression Vector -- 3. Overexpression and Purification of BphPs -- 4. Autophosphorylation -- 5. Phosphotransfer -- 6. Gel Mobility Shift Assay and DNase I Footprint -- 7. Gene Disruption -- 8. Analysis of the Photosynthetic Phenotypes of the BphP Mutants -- 9. Photochemical Measurements -- References -- Chapter 10: Biophysical Assays for Protein Interactions in the Wsp Sensory System and Biofilm Formation -- Abstract -- 1. Introduction -- 2. Analyses of the Shape and Molecular Weight of Proteins and Protein Complexes -- 3. Experimental Considerations -- 4. Case Studies -- 5. Concluding Remarks -- Acknowledgments -- References -- Chapter 11: High-Throughput Screening of Bacterial Protein Localization -- Abstract -- 1. Introduction -- 2. Pipeline Overview -- 3. Construction of a Caulobacter ORFeome -- 4. Construction of the Fluorescently Tagged Protein Library. , 5. Imaging the Caulobacter Protein Localization Library -- 6. Image Scoring and Analysis -- 7. Conclusion -- Acknowledgments -- References -- Chapter 12: In Vitro and In Vivo Analysis of the ArcB/A Redox Signaling Pathway -- Abstract -- 1. Introduction -- 2. In Vitro Characterization of the Arc TCS -- 3. In Vivo Characterization of the Arc TCS -- 4. Conclusions -- Acknowledgments -- References -- Chapter 13: Potassium Sensing Histidine Kinase in Bacillus subtilis -- Abstract -- 1. Introduction -- 2. Screen for Molecules that Stimulate KinC Sensor Kinase -- 3. Quantitative Analysis of the Activation of KinC -- 4. Structural Analysis of KinC -- 5. Monitoring the Signals Using Indirect Measurements -- 6. Applications of the System Signal-Kinase -- 7. Conclusions -- References -- Chapter 14: Two-Component Systems and Regulation of Developmental Progression in Myxococcus xanthus -- Abstract -- 1. Introduction -- 2. Generation of In-Frame Deletions or Point Mutations in the M. xanthus Genome -- 3. Phenotype Assays for Analysis of M. xanthus Development -- 4. Expression Analysis -- 5. In Vitro Biochemical Analysis of TCS Proteins -- Acknowledgments -- References -- Chapter 15: Two-Component Signaling to the Stress MAP Kinase Cascade in Fission Yeast -- Abstract -- 1. Introduction -- 2. Detection of Protein Interactions in the H2O2 Signaling Pathway -- 3. Detection of Cysteine S-Thiolation in Tdh1 GAPDH -- Acknowledgments -- References -- Chapter 16: Genetic and Biochemical Analysis of the SLN1 Pathway in Saccharomyces cerevisiae -- Abstract -- 1. Introduction -- 2. Materials and Methods -- Acknowledgments -- References -- Chapter 17: Analysis of Mitogen-Activated Protein Kinase Phosphorylation in Response to Stimulation of Histidine Kinase Signa -- Abstract -- 1. Introduction -- 2. Growth of Cultures and Exposure to Hyperosmotic Conditions or Fungicide. , 3. Mitogen-Activated Protein Kinase Assay -- 4. Adapting the MAPK Assay -- 5. Discussion -- Acknowledgments -- References -- Chapter 18: Biochemical Characterization of Plant Hormone Cytokinin-Receptor Histidine Kinases Using Microorganisms -- Abstract -- 1. Introduction -- 2. Characterization of Plant TCS Components in E. coli -- 3. Protocol for Histidine Kinase Assays in E. coli -- 4. Protocol of Cytokinin-Binding Assay with Intact E. coli Cells -- 5. Preparation of Radioactive Phospho-HPt Factor, and In Vitro Assay of Phosphotransfer to RR -- 6. Characterization of Plant TCS Components in S. cerevisiae -- 7. Protocol of Histidine Kinase Assay in S. cerevisiae -- 8. Protocol of HPt Factor Assay in S. cerevisiae -- 9. Protocol of Cytokinin-Binding Assay by Using S. pombe Membranes Enriched in AHK4/CRE1 -- Acknowledgments -- References -- Chapter 19: Characterization of Pseudo-Response Regulators in Plants -- Abstract -- 1. The Arabidopsis Circadian Clock -- 2. Detection of PRR Proteins -- 3. Localization of PRR Proteins -- 4. Exploring the Circadian Phenotypes of prr Mutants -- 5. Concluding Remarks -- Acknowledgments -- References -- Chapter 20: Reversible Histidine Phosphorylation in Mammalian Cells -- Abstract -- 1. Introduction -- 2. Analysis of Phosphorylation and Dephosphorylation of Histidine Residues In Vitro -- 3. Functional Analysis of NDPK/PHPT-1 Regulated Systems in Living Cells -- References -- Chapter 21: Histidine Phosphorylation in Histones and in Other Mammalian Proteins -- Abstract -- 1. Introduction -- 2. Chemical Phosphorylation of Histone H4 Proteins and Peptides -- 3. Detection of Phosphohistidine-Phosphoamino Acid Analysis -- 4. Filter-Based Assay of Alkali-Stable, Acid-Labile Protein Phosphorylation (Nytran Assay) -- 5. In-Gel Kinase Assay -- 6. Phosphorylation and Thiophosphorylation Site Analysis by Edman Sequencing. , 7. Mass Spectrometric Phosphopeptide Analysis -- Acknowledgments -- References -- Author Index -- Subject Index.