Two-Component Signaling Systems, Part B - 1st Edition - ISBN: 9780123738523, 9780080549460

Two-Component Signaling Systems, Part B, Volume 423

1st Edition

Serial Volume Editors: Melvin Simon Brian Crane Alexandrine Crane
eBook ISBN: 9780080549460
Hardcover ISBN: 9780123738523
Imprint: Academic Press
Published Date: 3rd July 2007
Page Count: 648
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Table of Contents

  • Contributors to Volume 423
    • Publisher Summary
  • Contents of Previous Volumes
  • 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
    • [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° Structure by Chemical Reactivity Scanning
      • Disulfide Mapping of Spatial Proximity and Conformational Changes
      • Disulfide Trapping of Thermal Backbone and Domain Motions
      • Acknowledgments
    • [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
    • [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
    • [5]: TonB/TolA Amino-Terminal Domain Modeling
      • Abstract
      • Introduction
      • Alanyl Replacement
      • TonB/TolA Chimeras
      • Acknowledgments
    • [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
    • [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
    • [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
    • [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
    • [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
    • [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
      • Acknowledgments
  • 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
    • [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
    • [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
    • [15]: Assays for CheC, FliY, and CheX as Representatives of Response Regulator Phosphatases
      • Abstract
      • Introduction
      • Assays
      • Phosphate Release Assay
      • Pulldowns
      • Acknowledgments
    • [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
  • 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
    • [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
    • [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
    • [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
    • [21]: Phenotypic Suppression Methods for Analyzing Intra- and Inter-Molecular Signaling Interactions of Chemoreceptors
      • Abstract
      • Introduction
      • Genetic Analyses of Chemoreceptors
      • Balancing Suppression: Methylation-Independent Chemoreceptors
      • Conformational Suppression within Receptor Molecules
      • Conformational Suppression Between Receptor Molecules
      • Acknowledgments
    • [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
      • Concluding Remarks
  • Section IV: Genome-Wide Analyses of Two-Component Systems
    • [23]: Two-Component Systems of Mycobacterium tuberculosis—Structure-Based Approaches
      • Abstract
      • Introduction
      • Orphan TCS Proteins
      • Information from Crystal Structures
      • Structural Genomics as a Driving Force
      • Domain Boundary Definitions
      • Protein Production as a Source of Material for Structural Studies and In Vitro Inhibition Assays
      • Crystallographic Studies
      • Information on Solution Structure from Small-Angle X-Ray Scattering
      • Structural Information Relating to Regulation Mechanisms
    • [24]: Transcriptomic Analysis of ArlRS Two-Component Signaling Regulon, a Global Regulator, in Staphylococcus aureus
      • Abstract
      • Introduction
      • Construction of an arlR Allelic Replacement Mutant in S. aureus
      • Purification of Total RNA From Wild Type and arlR Mutant Strains
      • cDNA Synthesis, cDNA Fragmentation, and Labeling
      • Microarray Analysis
      • Quantitative Real-Time RT-PCR Analysis
      • Acknowledgments
    • [25]: Global Analysis of Two-Component Gene Regulation in H. pylori by Mutation Analysis and Transcriptional Profiling
      • Abstract
      • Introduction
      • Functional Analysis of Essential Response Regulators of H. pylori
      • Characterization of the Regulons Controlled by the H. pylori Two-Component Systems
      • Design of the Experiment for Transcriptional Profiling
      • Validation of the Data
      • Concluding Remarks
    • [26]: Phosphotransfer Profiling: Systematic Mapping of Two-Component Signal Transduction Pathways and Phosphorelays
      • Abstract
      • Overview
      • Detailed Protocols
      • Interpretation and Analysis
      • Phosphorelays and Histidine Phosphotransferases
      • Concluding Remarks
    • [27]: Identification of Histidine Phosphorylations in Proteins Using Mass Spectrometry and Affinity-Based Techniques
      • Abstract
      • Introduction
      • Sample Fractionation
      • Phosphoprotein Enrichment
      • Gel Separation
      • Mass Spectrometry
      • Phosphopeptide Enrichment
      • Identification of Phosphohistidine in a Model Protein
      • Phosphorylation and Digestion of HPr
      • IMAC Conditions
      • MALDI-TOF MS Conditions
      • Enrichment of His-Phosphorylated Peptides
      • Selectivity for Phosphorylated Histidine
      • Detection of His-Phosphorylated Peptides
      • Specificity for Phosphohistidines
      • Differential Hydrolysis of Phosphohistidines
      • Summary and Conclusions
  • Subject Index
    • Publisher Summary
  • Author Index
    • Publisher Summary


Multicellular organisms must be able to adapt to cellular events to accommodate prevailing conditions. Sensory-response circuits operate by making use of a phosphorylation control mechanism known as the "two-component system."

Sections in Two-Component Signaling Systems, Part B include:

  • Structural Approaches
  • Reconstitution of Heterogeneous Systems
  • Intracellular Methods and Assays
  • Genome-Wide Analyses of Two-Component Systems

Key Features

  • Presents detailed protocols
  • Includes troubleshooting tips


Biochemists, geneticists, and molecular biologists.


No. of pages:
© Academic Press 2007
Academic Press
eBook ISBN:
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Ratings and Reviews

About the Serial Volume Editors

Melvin Simon Serial Volume Editor

Affiliations and Expertise

The Salk Institute, La Jolla, CA, USA

Brian Crane Serial Volume Editor

Affiliations and Expertise

Cornell University, Ithaca, NY, USA

Alexandrine Crane Serial Volume Editor

Affiliations and Expertise

Cornell University, Ithaca, NY, USA