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Two-Component Signaling Systems, Part B
1st Edition, Volume 423 - July 3, 2007
Editors: Melvin I. Simon, Brian Crane, Alexandrine Crane
Language: English
Hardback ISBN:9780123738523
9 7 8 - 0 - 1 2 - 3 7 3 8 5 2 - 3
eBook ISBN:9780080549460
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
Presents detailed protocols
Includes troubleshooting tips
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
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