Two-Component Signaling Systems, Part B

1st Edition, Volume 423 - July 3, 2007
Latest edition
Editors: Melvin I. Simon, Brian Crane, Alexandrine Crane
Language: English
Hardback ISBN:
9 7 8 - 0 - 1 2 - 3 7 3 8 5 2 - 3
eBook ISBN:
9 7 8 - 0 - 0 8 - 0 5 4 9 4 6 - 0

Multicellular organisms must be able to adapt to cellular events to accommodate prevailing conditions. Sensory-response circuits operate by making use of a phosphorylation co… Read more

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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

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 LuxPQ_p
  - Purification of LuxP, LuxQ_p, and LuxPQ_p
  - Crystallization of LuxPQ_p 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

Life Sciences

Physical Sciences & Engineering

Social Sciences & Humanities

Health

Two-Component Signaling Systems, Part B

Fuel your confidence!

Melvin I. Simon

Brian Crane

Alexandrine Crane

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