Elsevier S & T

Two-Component Signaling Systems, Part B

Simon, Melvin I.

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Table of contents

Copyright Pageiv
Table of Contentsv
Contributors to Volume 423ix
Volume in Seriesxiii
Section I: Structural Approaches1
Chapter 1: The PICM Chemical Scanning Method for Identifying Domain-Domain and Protein-Protein Inter3
Introduction4
Comparison of the PICM Method with Other Scanning Approaches4
PICM Studies of the Core Signaling Complex of Bacterial Chemotaxis6
Generalizing the PICM Method to Map Docking Sites in Other Systems7
Incorporation of an Affinity Tag and Creation of a Cysless Protein8
Choice of Positions for Cys Incorporation and Creation of a Mutant Library10
Selection of a Cys-Specific Probe for Chemical Modification12
Probe Labeling and Purification of the Single Cys Mutants14
Quantitation of Probe Coupling16
Measuring Functional Effects of Cys Substitution and Bulky Probe Coupling18
Interpretation of Results-Mapping Out Docking Sites20
Acknowledgments22
References22
Chapter 2: Use of Site-Directed Cysteine and Disulfide Chemistry to Probe Protein Structure and Dyna25
Introduction26
Site-Directed Cysteine and Disulfide Chemistry: History26
Site-Directed Cysteine and Disulfide Chemistry: Applications and Limitations28
Incorporation of an Affinity Tag and Creation of a Cysless Protein29
Choice of Positions for Cys Incorporation and Creation of a Mutant Library30
Analysis of 2degStructure by Chemical Reactivity Scanning31
Disulfide Mapping of Spatial Proximity and Conformational Changes36
Disulfide Trapping of Thermal Backbone and Domain Motions45
Acknowledgments49
References49
Chapter 3: Measuring Distances by Pulsed Dipolar ESR Spectroscopy: Spin-Labeled Histidine Kinases52
Introduction52
Dipolar ESR Spectroscopy55
Case Study: PDS Reconstruction of Histidine Kinases Signaling Complex87
Concluding Remarks108
Acknowledgments108
References108
Chapter 4: Rigid Body Refinement of Protein Complexes with Long-Range Distance Restraints from Pulse117
Introduction117
Method118
Initial Conformation of the Complex119
Results120
Discussion129
Acknowledgments132
References132
Chapter 5: TonB/TolA Amino-Terminal Domain Modeling134
Introduction134
Alanyl Replacement138
TonB/TolA Chimeras141
Acknowledgments147
References147
Chapter 6: Functional Dynamics of Response Regulators Using NMR Relaxation Techniques149
Introduction149
The Experimental Setup151
Two-State Allosteric Activation Identified by NMR Chemical Shift Analysis153
Two-State Allosteric Activation Buttressed by Standard NMR Relaxation Experiments154
A New Approach for Quantitative Analysis of Microsecond Protein Dynamics156
Conclusions161
Acknowledgments162
References162
Chapter 7: The Design and Development of Tar-EnvZ Chimeric Receptors166
Introduction166
Construction of Tar-EnvZ Chimeric Protein, Taz168
Asp-Dependent Induction of ompC-lacZ Fusion Gene by Taz and OmpR169
Phenotype Analysis of the Taz Construct171
Regulation of Binding of Asp to One of Two Asp-Binding Pockets of Tar Receptor to Study Signal Trans172
The Right Configuration of HAMP Domain is Crucial for Proper Signal Transduction in a Tar-EnvZ Chime177
Conclusions180
Acknowledgments180
References180
Chapter 8: Functional and Structural Characterization of EnvZ, an Osmosensing Histidine Kinase of E.184
Introduction184
Expression and Purification of EnvZc187
Expression and Purification of Domain A and Domain B188
Characterization of EnvZc189
Characterization of EnvZ with Help of its Specific Mutants194
Creation of a Monomeric Histidine Kinase Using EnvZc198
NMR Structural Analysis of Domain A and Domain B199
Conclusion200
Acknowledgments200
References200
Chapter 9: Light Modulation of Histidine-Kinase Activity in Bacterial Phytochromes Monitored by Size203
Introduction203
Sample Preparation206
Photoconversion, Experimental Light Conditions, Protein Concentration208
Size Exclusion Chromatography210
Protein Crosslinking213
Limited Proteolysis216
Autophosphorylation217
References219
Chapter 10: A Temperature-Sensing Histidine Kinase-Function, Genetics, and Membrane Topology222
Introduction222
Genetic Approaches to Characterize CorRSP225
Transcriptional Analysis227
Biochemical Characterization of CorRSP231
Topological Analysis of the HPK CorS235
Concluding Remarks243
Acknowledgments245
References245
Chapter 11: The Regulation of Histidine Sensor Kinase Complexes by Quorum Sensing Signal Molecules250
Introduction250
Bacterial Quorum Sensing251
The V. harveyi AI-2 Signal Transduction Pathway251
Regulation of the LuxPQ Receptor Complex by AI-2253
Expression of Wild-Type and Mutant LuxPQp254
Purification of LuxP, LuxQp, and LuxPQp256
Crystallization of LuxPQp Complexes258
Functional Analysis260
Conclusions261
Acknowledgments262
References262
Section II: Reconstitution of Heterogeneous Systems265
Chapter 12: Liposome-Mediated Assembly of Receptor Signaling Complexes267
Introduction267
Results-Biochemical Activity of Liposome-Assembled Receptor Fragments272
Methods287
Conclusion293
Acknowledgment294
References294
Chapter 13: Analyzing Transmembrane Chemoreceptors Using In Vivo Disulfide Formation Between Introdu299
Introduction299
Disulfide Formation In Vivo: Applications and Limitations300
Oxidation Reagents303
Oxidation Treatments That Preserve In Vivo Function305
Experimental Designs306
Procedures310
Analysis313
Closing Comments314
Acknowledgments315
References315
Chapter 14: Using Nanodiscs to Create Water-Soluble Transmembrane Chemoreceptors Inserted in Lipid B317
Introduction317
Developing a Protocol for Producing Nanodisc-Embedded Protein319
Preparation of Nanodisc-Embedded Chemoreceptor325
Preparation of Cytoplasmic Membranes with High Tar-6H Content329
Receptor Purification330
Preparation of Receptor-Containing Nanodiscs331
Analysis of Receptor-Containing Nanodiscs334
Acknowledgments334
References334
Chapter 15: Assays for CheC, FliY, and CheX as Representatives of Response Regulator Phosphatases336
Introduction336
Assays339
Phosphate Release Assay341
Pulldowns344
Acknowledgments347
References347
Chapter 16: Genetic Dissection of Signaling Through the Rcs Phosphorelay349
Overview349
Flowchart of Testing: Signaling Inputs350
Analysis of the Regulation of a Target Gene351
Analysis of Signaling via the Rcs Phosphorelay355
RcsC-Dependent Signaling358
RcsA-Dependent Signaling: Increased RcsA Synthesis or Stability358
Determining Whether a Strain Carries a lon Mutation or is Phenotypically Lon-359
Conclusions360
Acknowledgments360
References360
Section III: Intracellular Methods and Assays363
Chapter 17: In Vivo Measurement by FRET of Pathway Activity in Bacterial Chemotaxis365
Introduction365
FRET367
FRET Measurement of the Interaction Between CheY-YFP and CheZ-CFP in a Population of Bacteria Fixed368
FRET Measurement of the Interaction Between CheY-YFP and CheZ-CFP in Single Bacteria Fixed to a Micr377
BRET Measurement of the Interaction Between YFP-CheY and-CheZ-RLUC in a Population of Bacteria Swimm382
Comparison of Different Approaches and Application to Other Two-Component Systems387
References389
Chapter 18: In Vivo and In Vitro Analysis of the Rhodobacter sphaeroides Chemotaxis Signaling Comple392
Introduction392
In Vitro Analysis of Signaling by the Kinase Cluster395
Genomic Replacements with Fluorescent Protein Fusions for Studying Protein Localization404
Assessing the Functionality of the Fluorescent Protein Fusions410
Summary411
References411
Chapter 19: In Vivo Crosslinking Methods for Analyzing the Assembly and Architecture of Chemorecepto414
Introduction414
Use of a Lysine-Targeted Crosslinker to Probe Receptor-Receptor Interactions in Cells416
Use of Cys-Targeted Crosslinking to Probe for the Trimer-of -Dimers Geometry in Cellular Chemorecept418
Intracytoplasmic Disulfide Crosslinks419
A Trifunctional Cys-Targeted Crosslinker422
TMEA Competition Assay: A Tool for Assessing the Trimer-Forming Ability of Mutant Receptors425
Exchange Assay: Dynamic Changes in Trimer Composition as a Consequence of Changes in the Receptor Po427
Concluding Remarks428
Acknowledgments429
References429
Chapter 20: A "Bucket of Light " for Viewing Bacterial Colonies in Soft Agar432
Viewing Colonies Grown in Soft Agar432
Building a Bucket of Light433
Acknowledgments435
References435
Chapter 21: Phenotypic Suppression Methods for Analyzing Intra- and Inter-Molecular Signaling Intera436
Introduction436
Genetic Analyses of Chemoreceptors439
Balancing Suppression: Methylation-Independent Chemoreceptors443
Conformational Suppression within Receptor Molecules446
Conformational Suppression Between Receptor Molecules451
Acknowledgments455
References455
Chapter 22: Single-Cell Analysis of Gene Expression by Fluorescence Microscopy458
Introduction458
Transcriptional Reporters459
Measuring Cellular Fluorescence by Microscopy464
Agarose Pads465
Fluorescence Microscopy and Image Acquisition467
Image Analysis469
Concluding Remarks473
References474
Section IV: Genome-Wide Analyses of Two-Component Systems477
Chapter 23: Two-Component Systems of Mycobacterium tuberculosis-Structure-Based Approaches479
Introduction479
Orphan TCS Proteins483
Information from Crystal Structures485
Structural Genomics as a Driving Force485
Domain Boundary Definitions486
Protein Production as a Source of Material for Structural Studies and In Vitro Inhibition Assays486
Crystallographic Studies490
Information on Solution Structure from Small-Angle X-Ray Scattering491
Structural Information Relating to Regulation Mechanisms496
References497
Chapter 24: Transcriptomic Analysis of ArlRS Two-Component Signaling Regulon, a Global Regulator, in502
Introduction502
Construction of an arlR Allelic Replacement Mutant in S. aureus504
Purification of Total RNA From Wild Type and arlR Mutant Strains505
cDNA Synthesis, cDNA Fragmentation, and Labeling506
Microarray Analysis508
Quantitative Real-Time RT-PCR Analysis510
Acknowledgments512
References512
Chapter 25: Global Analysis of Two-Component Gene Regulation in H. pylori by Mutation Analysis and T514
Introduction514
Functional Analysis of Essential Response Regulators of H. pylori517
Characterization of the Regulons Controlled by the H. pylori Two-Component Systems520
Design of the Experiment for Transcriptional Profiling522
Validation of the Data526
Concluding Remarks527
References527
Chapter 26: Phosphotransfer Profiling: Systematic Mapping of Two-Component Signal Transduction Pathw531
Overview531
Detailed Protocols536
Interpretation and Analysis542
Phosphorelays and Histidine Phosphotransferases544
Concluding Remarks547
References547
Chapter 27: Identification of Histidine Phosphorylations in Proteins Using Mass Spectrometry and Aff549
Introduction549
Sample Fractionation551
Phosphoprotein Enrichment553
Gel Separation554
Mass Spectrometry556
Phosphopeptide Enrichment559
Identification of Phosphohistidine in a Model Protein561
Phosphorylation and Digestion of HPr561
IMAC Conditions562
MALDI-TOF MS Conditions562
Enrichment of His-Phosphorylated Peptides563
Selectivity for Phosphorylated Histidine564
Detection of His-Phosphorylated Peptides565
Specificity for Phosphohistidines567
Differential Hydrolysis of Phosphohistidines567
Summary and Conclusions568
References569
Author Index573
Subject Index599

Book details

Vendor Elsevier S & T
SKU 9780123738523
ISBN-13 9780080549460
Author Simon, Melvin I.
Category Science
Subject Molecular Biology

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

Presents detailed protocols
Includes troubleshooting tips

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