Crane Two-Component Signaling Systems, Part B

1. Auflage 2007
ISBN: 978-0-08-054946-0
Verlag: Elsevier Science & Techn.
Format: EPUB
Kopierschutz: 6 - ePub Watermark

E-Book, Englisch, Band 423, 648 Seiten

Reihe: Methods in Enzymology

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

Autoren/Hrsg.

Crane, Brian

Crane, Alexandrine

Inhaltsverzeichnis

1;Cover;1
2;Copyright Page;5
3;Table of Contents;6
4;Contributors to Volume 423;10
5;Volume in Series;14
6;Section I: Structural Approaches;38
6.1;Chapter 1: The PICM Chemical Scanning Method for Identifying Domain-Domain and Protein-Protein Interfaces: Applications to the Core Signaling Complex of E.Coli Chemotaxis;40
6.1.1;Introduction;41
6.1.2;Comparison of the PICM Method with Other Scanning Approaches;41
6.1.3;PICM Studies of the Core Signaling Complex of Bacterial Chemotaxis;43
6.1.4;Generalizing the PICM Method to Map Docking Sites in Other Systems;44
6.1.5;Incorporation of an Affinity Tag and Creation of a Cysless Protein;45
6.1.6;Choice of Positions for Cys Incorporation and Creation of a Mutant Library;47
6.1.7;Selection of a Cys-Specific Probe for Chemical Modification;49
6.1.8;Probe Labeling and Purification of the Single Cys Mutants;51
6.1.9;Quantitation of Probe Coupling;53
6.1.10;Measuring Functional Effects of Cys Substitution and Bulky Probe Coupling;55
6.1.11;Interpretation of Results-Mapping Out Docking Sites;57
6.1.12;Acknowledgments;59
6.1.13;References;59
6.2;Chapter 2: Use of Site-Directed Cysteine and Disulfide Chemistry to Probe Protein Structure and Dynamics: Applications to Soluble and Transmembrane Receptors of Bacterial Chemotaxis;62
6.2.1;Introduction;63
6.2.2;Site-Directed Cysteine and Disulfide Chemistry: History;63
6.2.3;Site-Directed Cysteine and Disulfide Chemistry: Applications and Limitations;65
6.2.4;Incorporation of an Affinity Tag and Creation of a Cysless Protein;66
6.2.5;Choice of Positions for Cys Incorporation and Creation of a Mutant Library;67
6.2.6;Analysis of 2degStructure by Chemical Reactivity Scanning;68
6.2.7;Disulfide Mapping of Spatial Proximity and Conformational Changes;73
6.2.8;Disulfide Trapping of Thermal Backbone and Domain Motions;82
6.2.9;Acknowledgments;86
6.2.10;References;86
6.3;Chapter 3: Measuring Distances by Pulsed Dipolar ESR Spectroscopy: Spin-Labeled Histidine Kinases;89
6.3.1;Introduction;89
6.3.2;Dipolar ESR Spectroscopy;92
6.3.3;Case Study: PDS Reconstruction of Histidine Kinases Signaling Complex;124
6.3.4;Concluding Remarks;145
6.3.5;Acknowledgments;145
6.3.6;References;145
6.4;Chapter 4: Rigid Body Refinement of Protein Complexes with Long-Range Distance Restraints from Pulsed Dipolar ESR;154
6.4.1;Introduction;154
6.4.2;Method;155
6.4.3;Initial Conformation of the Complex;156
6.4.4;Results;157
6.4.5;Discussion;166
6.4.6;Acknowledgments;169
6.4.7;References;169
6.5;Chapter 5: TonB/TolA Amino-Terminal Domain Modeling;171
6.5.1;Introduction;171
6.5.2;Alanyl Replacement;175
6.5.3;TonB/TolA Chimeras;178
6.5.4;Acknowledgments;184
6.5.5;References;184
6.6;Chapter 6: Functional Dynamics of Response Regulators Using NMR Relaxation Techniques;186
6.6.1;Introduction;186
6.6.2;The Experimental Setup;188
6.6.3;Two-State Allosteric Activation Identified by NMR Chemical Shift Analysis;190
6.6.4;Two-State Allosteric Activation Buttressed by Standard NMR Relaxation Experiments;191
6.6.5;A New Approach for Quantitative Analysis of Microsecond Protein Dynamics;193
6.6.6;Conclusions;198
6.6.7;Acknowledgments;199
6.6.8;References;199
6.7;Chapter 7: The Design and Development of Tar-EnvZ Chimeric Receptors;203
6.7.1;Introduction;203
6.7.2;Construction of Tar-EnvZ Chimeric Protein, Taz;205
6.7.3;Asp-Dependent Induction of ompC-lacZ Fusion Gene by Taz and OmpR;206
6.7.4;Phenotype Analysis of the Taz Construct;208
6.7.5;Regulation of Binding of Asp to One of Two Asp-Binding Pockets of Tar Receptor to Study Signal Transduction;209
6.7.6;The Right Configuration of HAMP Domain is Crucial for Proper Signal Transduction in a Tar-EnvZ Chimeric Protein;214
6.7.7;Conclusions;217
6.7.8;Acknowledgments;217
6.7.9;References;217
6.8;Chapter 8: Functional and Structural Characterization of EnvZ, an Osmosensing Histidine Kinase of E. coli;221
6.8.1;Introduction;221
6.8.2;Expression and Purification of EnvZc;224
6.8.3;Expression and Purification of Domain A and Domain B;225
6.8.4;Characterization of EnvZc;226
6.8.5;Characterization of EnvZ with Help of its Specific Mutants;231
6.8.6;Creation of a Monomeric Histidine Kinase Using EnvZc;235
6.8.7;NMR Structural Analysis of Domain A and Domain B;236
6.8.8;Conclusion;237
6.8.9;Acknowledgments;237
6.8.10;References;237
6.9;Chapter 9: Light Modulation of Histidine-Kinase Activity in Bacterial Phytochromes Monitored by Size Exclusion Chromatography, Crosslinking, and Limited Proteolysis;240
6.9.1;Introduction;240
6.9.2;Sample Preparation;243
6.9.3;Photoconversion, Experimental Light Conditions, Protein Concentration;245
6.9.4;Size Exclusion Chromatography;247
6.9.5;Protein Crosslinking;250
6.9.6;Limited Proteolysis;253
6.9.7;Autophosphorylation;254
6.9.8;References;256
6.10;Chapter 10: A Temperature-Sensing Histidine Kinase-Function, Genetics, and Membrane Topology;259
6.10.1;Introduction;259
6.10.2;Genetic Approaches to Characterize CorRSP;262
6.10.3;Transcriptional Analysis;264
6.10.4;Biochemical Characterization of CorRSP;268
6.10.5;Topological Analysis of the HPK CorS;272
6.10.6;Concluding Remarks;280
6.10.7;Acknowledgments;282
6.10.8;References;282
6.11;Chapter 11: The Regulation of Histidine Sensor Kinase Complexes by Quorum Sensing Signal Molecules;287
6.11.1;Introduction;287
6.11.2;Bacterial Quorum Sensing;288
6.11.3;The V. harveyi AI-2 Signal Transduction Pathway;288
6.11.4;Regulation of the LuxPQ Receptor Complex by AI-2;290
6.11.5;Expression of Wild-Type and Mutant LuxPQp;291
6.11.6;Purification of LuxP, LuxQp, and LuxPQp;293
6.11.7;Crystallization of LuxPQp Complexes;295
6.11.8;Functional Analysis;297
6.11.9;Conclusions;298
6.11.10;Acknowledgments;299
6.11.11;References;299
7;Section II: Reconstitution of Heterogeneous Systems;302
7.1;Chapter 12: Liposome-Mediated Assembly of Receptor Signaling Complexes;304
7.1.1;Introduction;304
7.1.2;Results-Biochemical Activity of Liposome-Assembled Receptor Fragments;309
7.1.3;Methods;324
7.1.4;Conclusion;330
7.1.5;Acknowledgment;331
7.1.6;References;331
7.2;Chapter 13: Analyzing Transmembrane Chemoreceptors Using In Vivo Disulfide Formation Between Introduced Cysteines;336
7.2.1;Introduction;336
7.2.2;Disulfide Formation In Vivo: Applications and Limitations;337
7.2.3;Oxidation Reagents;340
7.2.4;Oxidation Treatments That Preserve In Vivo Function;342
7.2.5;Experimental Designs;343
7.2.6;Procedures;347
7.2.7;Analysis;350
7.2.8;Closing Comments;351
7.2.9;Acknowledgments;352
7.2.10;References;352
7.3;Chapter 14: Using Nanodiscs to Create Water-Soluble Transmembrane Chemoreceptors Inserted in Lipid Bilayers;354
7.3.1;Introduction;354
7.3.2;Developing a Protocol for Producing Nanodisc-Embedded Protein;356
7.3.3;Preparation of Nanodisc-Embedded Chemoreceptor;362
7.3.4;Preparation of Cytoplasmic Membranes with High Tar-6H Content;366
7.3.5;Receptor Purification;367
7.3.6;Preparation of Receptor-Containing Nanodiscs;368
7.3.7;Analysis of Receptor-Containing Nanodiscs;371
7.3.8;Acknowledgments;371
7.3.9;References;371
7.4;Chapter 15: Assays for CheC, FliY, and CheX as Representatives of Response Regulator Phosphatases;373
7.4.1;Introduction;373
7.4.2;Assays;376
7.4.3;Phosphate Release Assay;378
7.4.4;Pulldowns;381
7.4.5;Acknowledgments;384
7.4.6;References;384
7.5;Chapter 16: Genetic Dissection of Signaling Through the Rcs Phosphorelay;386
7.5.1;Overview;386
7.5.2;Flowchart of Testing: Signaling Inputs;387
7.5.3;Analysis of the Regulation of a Target Gene;388
7.5.4;Analysis of Signaling via the Rcs Phosphorelay;392
7.5.5;RcsC-Dependent Signaling;395
7.5.6;RcsA-Dependent Signaling: Increased RcsA Synthesis or Stability;395
7.5.7;Determining Whether a Strain Carries a lon Mutation or is Phenotypically Lon-;396
7.5.8;Conclusions;397
7.5.9;Acknowledgments;397
7.5.10;References;397
8;Section III: Intracellular Methods and Assays;400
8.1;Chapter 17: In Vivo Measurement by FRET of Pathway Activity in Bacterial Chemotaxis;402
8.1.1;Introduction;402
8.1.2;FRET;404
8.1.3;FRET Measurement of the Interaction Between CheY-YFP and CheZ-CFP in a Population of Bacteria Fixed to a Microscope Cover Slip;405
8.1.4;FRET Measurement of the Interaction Between CheY-YFP and CheZ-CFP in Single Bacteria Fixed to a Microscope Cover Slip;414
8.1.5;BRET Measurement of the Interaction Between YFP-CheY and-CheZ-RLUC in a Population of Bacteria Swimming in a Cuvette;419
8.1.6;Comparison of Different Approaches and Application to Other Two-Component Systems;424
8.1.7;References;426
8.2;Chapter 18: In Vivo and In Vitro Analysis of the Rhodobacter sphaeroides Chemotaxis Signaling Complexes;429
8.2.1;Introduction;429
8.2.2;In Vitro Analysis of Signaling by the Kinase Cluster;432
8.2.3;Genomic Replacements with Fluorescent Protein Fusions for Studying Protein Localization;441
8.2.4;Assessing the Functionality of the Fluorescent Protein Fusions;447
8.2.5;Summary;448
8.2.6;References;448
8.3;Chapter 19: In Vivo Crosslinking Methods for Analyzing the Assembly and Architecture of Chemoreceptor Arrays;451
8.3.1;Introduction;451
8.3.2;Use of a Lysine-Targeted Crosslinker to Probe Receptor-Receptor Interactions in Cells;453
8.3.3;Use of Cys-Targeted Crosslinking to Probe for the Trimer-of -Dimers Geometry in Cellular Chemoreceptor Assemblies;455
8.3.4;Intracytoplasmic Disulfide Crosslinks;456
8.3.5;A Trifunctional Cys-Targeted Crosslinker;459
8.3.6;TMEA Competition Assay: A Tool for Assessing the Trimer-Forming Ability of Mutant Receptors;462
8.3.7;Exchange Assay: Dynamic Changes in Trimer Composition as a Consequence of Changes in the Receptor Population;464
8.3.8;Concluding Remarks;465
8.3.9;Acknowledgments;466
8.3.10;References;466
8.4;Chapter 20: A "Bucket of Light " for Viewing Bacterial Colonies in Soft Agar;469
8.4.1;Viewing Colonies Grown in Soft Agar;469
8.4.2;Building a Bucket of Light;470
8.4.3;Acknowledgments;472
8.4.4;References;472
8.5;Chapter 21: Phenotypic Suppression Methods for Analyzing Intra- and Inter-Molecular Signaling Interactions of Chemoreceptors;473
8.5.1;Introduction;473
8.5.2;Genetic Analyses of Chemoreceptors;476
8.5.3;Balancing Suppression: Methylation-Independent Chemoreceptors;480
8.5.4;Conformational Suppression within Receptor Molecules;483
8.5.5;Conformational Suppression Between Receptor Molecules;488
8.5.6;Acknowledgments;492
8.5.7;References;492
8.6;Chapter 22: Single-Cell Analysis of Gene Expression by Fluorescence Microscopy;495
8.6.1;Introduction;495
8.6.2;Transcriptional Reporters;496
8.6.3;Measuring Cellular Fluorescence by Microscopy;501
8.6.4;Agarose Pads;502
8.6.5;Fluorescence Microscopy and Image Acquisition;504
8.6.6;Image Analysis;506
8.6.7;Concluding Remarks;510
8.6.8;References;511
9;Section IV: Genome-Wide Analyses of Two-Component Systems;514
9.1;Chapter 23: Two-Component Systems of Mycobacterium tuberculosis-Structure-Based Approaches;516
9.1.1;Introduction;516
9.1.2;Orphan TCS Proteins;520
9.1.3;Information from Crystal Structures;522
9.1.4;Structural Genomics as a Driving Force;522
9.1.5;Domain Boundary Definitions;523
9.1.6;Protein Production as a Source of Material for Structural Studies and In Vitro Inhibition Assays;523
9.1.7;Crystallographic Studies;527
9.1.8;Information on Solution Structure from Small-Angle X-Ray Scattering;528
9.1.9;Structural Information Relating to Regulation Mechanisms;533
9.1.10;References;534
9.2;Chapter 24: Transcriptomic Analysis of ArlRS Two-Component Signaling Regulon, a Global Regulator, in Staphylococcus aureus;539
9.2.1;Introduction;539
9.2.2;Construction of an arlR Allelic Replacement Mutant in S. aureus;541
9.2.3;Purification of Total RNA From Wild Type and arlR Mutant Strains;542
9.2.4;cDNA Synthesis, cDNA Fragmentation, and Labeling;543
9.2.5;Microarray Analysis;545
9.2.6;Quantitative Real-Time RT-PCR Analysis;547
9.2.7;Acknowledgments;549
9.2.8;References;549
9.3;Chapter 25: Global Analysis of Two-Component Gene Regulation in H. pylori by Mutation Analysis and Transcriptional Profiling;551
9.3.1;Introduction;551
9.3.2;Functional Analysis of Essential Response Regulators of H. pylori;554
9.3.3;Characterization of the Regulons Controlled by the H. pylori Two-Component Systems;557
9.3.4;Design of the Experiment for Transcriptional Profiling;559
9.3.5;Validation of the Data;563
9.3.6;Concluding Remarks;564
9.3.7;References;564
9.4;Chapter 26: Phosphotransfer Profiling: Systematic Mapping of Two-Component Signal Transduction Pathways and Phosphorelays;568
9.4.1;Overview;568
9.4.2;Detailed Protocols;573
9.4.3;Interpretation and Analysis;579
9.4.4;Phosphorelays and Histidine Phosphotransferases;581
9.4.5;Concluding Remarks;584
9.4.6;References;584
9.5;Chapter 27: Identification of Histidine Phosphorylations in Proteins Using Mass Spectrometry and Affinity-Based Techniques;586
9.5.1;Introduction;586
9.5.2;Sample Fractionation;588
9.5.3;Phosphoprotein Enrichment;590
9.5.4;Gel Separation;591
9.5.5;Mass Spectrometry;593
9.5.6;Phosphopeptide Enrichment;596
9.5.7;Identification of Phosphohistidine in a Model Protein;598
9.5.8;Phosphorylation and Digestion of HPr;598
9.5.9;IMAC Conditions;599
9.5.10;MALDI-TOF MS Conditions;599
9.5.11;Enrichment of His-Phosphorylated Peptides;600
9.5.12;Selectivity for Phosphorylated Histidine;601
9.5.13;Detection of His-Phosphorylated Peptides;602
9.5.14;Specificity for Phosphohistidines;604
9.5.15;Differential Hydrolysis of Phosphohistidines;604
9.5.16;Summary and Conclusions;605
9.5.17;References;606
10;Author Index;610
11;Subject Index;636

Leseproben

[1] The PICM Chemical Scanning Method for Identifying Domain–Domain and Protein–Protein Interfaces: Applications to the Core Signaling Complex of E. coli Chemotaxis
Randal B. Bass; Aaron S. Miller; Susan L. Gloor; Joseph J. Falke Abstract
The number of known protein structures is growing exponentially (Berman et al., 2000), but the structural mapping of essential domain–domain and protein–protein interaction surfaces has advanced more slowly. It is particularly difficult to analyze the interaction surfaces of membrane proteins on a structural level, both because membrane proteins are less accessible to high-resolution structural analysis and because the membrane environment is often required for native complex formation. The Protein-Interactions-by-Cysteine-Modification (PICM) method is a generalizable, in vitro chemical scanning approach that can be applied to many protein complexes, in both membrane-bound and soluble systems. The method begins by engineering Cys residues on the surface of a protein of known structure, then a bulky probe is coupled to each Cys residue. Next, the effects of both Cys substitution and bulky probe attachment are measured on the assembly and the activity of the target complex. Bulky probe coupling at an essential docking site disrupts complex assembly and/or activity, while coupling outside the site typically has little or no effect. PICM has been successfully applied to the core signaling complex of the E. coli and S. typhimurium chemotaxis pathway, where it has mapped out essential docking surfaces on transmembrane chemoreceptor (Tar) and histidine kinase (CheA) components (Bass and Falke, 1998; Mehan et al., 2003; Miller et al., 2006). The approach shares similarities with other important scanning methods like alanine and tryptophan scanning (Cunningham and Wells, 1989; Sharp et al., 1995a), but has two unique features: (1) functional effects are determined for both small volume (Cys) and large volume (bulky probe) side chain substitutions in the same experiment, and (2) nonperturbing positions are identified at which Cys residues and bulky probes can be introduced for subsequent biochemical and biophysical studies, without significant effects on complex assembly or activity. Introduction
Domain–domain and protein–protein interactions are essential to the functions of many, if not most, proteins. Such molecular contacts are especially crucial in signaling pathways, including the two-component signaling pathways of prokaryotic organisms. Signal transduction through a cellular circuit typically requires both intramolecular and intermolecular contacts. Domain–domain interactions within a single pathway component are often essential for the transmission of internal conformational signals, while protein–protein interactions between pathway components are needed for transmission of information throughout the cellular circuit. Thus, a molecular understanding of signal transduction requires methods capable of mapping and analyzing domain–domain and protein–protein contacts. More generally, such mapping methods can provide useful information about a wide array of cellular processes involving interactions between domains or different proteins, ranging from interdomain allostery within enzymes, to cooperative interactions between the subunits of homo-oligomers, to the assembly and regulation of multiprotein complexes. Comparison of the PICM Method with Other Scanning Approaches
Several scanning methods have proven useful in analyzing the location, function, and physical–chemical parameters of protein interaction surfaces, including alanine scanning, tryptophan scanning, and the Protein-Interactions-by-Cysteine-Modifications (PICM) method (Bass and Falke, 1998; Cunningham and Wells, 1989; Mehan et al., 2003; Miller et al., 2006; Sharp et al., 1995a). These methods are most useful when they are applied to proteins of known structure, so that engineered mutations can be targeted exclusively to the protein surface where effects on the native fold of the modified protein are minimal, while effects on the docking interaction are maximal. The methods can all be applied effectively to soluble proteins, but, unlike many other structural methods, are also useful in the analysis of membrane proteins even in their native bilayer environments. Alanine scanning substitutes Ala at selected surface positions, then measures the effects of each Ala substitution on the affinity of the docking interaction or on the activity of the docked complex (Cunningham and Wells, 1989; Wells, 1996). With the sole exception of Gly positions, substitution of Ala for a docking site residue truncates a larger side chain while having minimal effects on backbone flexibility. To a first approximation, then, the resulting effect of Ala substitution on docking affinity or activity reveals the contribution of an individual native side chain to the docking interaction. Alanine scanning is often used to analyze the physical chemistry of docking when the structure of the assembled complex is already known, but it can also map out the location of an unknown docking site on the surface of an isolated domain or protein as long as its docking partner is available for affinity and/or activity studies. Tryptophan scanning substitutes Trp at selected surface positions, then determines the effect of each Trp substitution on complex assembly or activity (Sharp et al., 1995a,b). Since Trp is the largest natural side chain, this substitution always increases side chain volume, thereby maximizing the probability of a dramatic effect on docking affinity. It follows that Trp substitutions within a docking site will generally yield measurable perturbations, making Trp scanning an efficient method of mapping out unknown docking sites on protein surfaces. For protein surfaces buried within a membrane bilayer, Trp scanning is particularly useful because the membrane environment often prevents the covalent coupling of extrinsic bulky probes, thereby largely eliminating the use of extrinsic probes in mapping the docking site. The PICM method is complementary to the alanine and tryptophan scanning approaches, combining some of their strengths and offering additional advantages, particularly in systems where further biochemical and biophysical studies of the purified components are planned. PICM makes use of the unique chemical and physical properties of the Cys side chain (Bass and Falke, 1999; Falke et al., 1986), which the method introduces at water-exposed surface positions scattered throughout the region where an unmapped docking site could reside. Subsequently, these engineered Cys residues are covalently modified with a bulky probe, and the effects of both the Cys substitution and bulky probe modification are determined on complex activity and assembly (Bass and Falke, 1998; Mehan et al., 2003; Miller et al., 2006). The engineered Cys side chain is smaller than all others except Gly, Ala, and Ser. Thus, Cys substitution typically replaces a native residue with a smaller side chain much as alanine scanning does. The bulky probe chosen for subsequent coupling to the Cys side chain is significantly larger than tryptophan, and thus replaces all native residues with a larger side chain that yields even better disruption of docking interactions than does tryptophan scanning. It follows that the PICM approach simultaneously determines the effects of smaller and larger side chain substitutions at most positions. Moreover, the PICM method yields an optimized labeling library of non-perturbing positions at which Cys substitution and bulky probe incorporation have little or no effect on docking interactions and activity. Such a library is of great utility in further biochemical and biophysical studies requiring sulfhydryl chemistry or the attachment of large spectroscopic probes or crosslinkers. In practice, the PICM approach is limited primarily to the analysis of water-exposed docking sites on purified proteins or on the extracellular domains of proteins in living cells. Because the PICM method requires coupling of a bulky probe to a Cys residue, typically via an alkylation or disulfide exchange reaction involving the Cys sulfanion, the approach is easily applied to aqueous docking sites accessible to the probe. By contrast, the PICM method is less useful for (a) lipid-exposed docking sites where the coupling reaction proceeds slowly because the low dielectric environment raises the sulfhydryl pKa, and (b) cytoplasmically exposed docking sites in living cells where the plasma membrane barrier and high cytoplasmic glutathione concentration typically interfere with probe coupling. In such cases, alanine and tryptophan scanning methods are generally preferred. PICM Studies of the Core Signaling Complex of Bacterial Chemotaxis
The PICM method was originally developed in studies of the core signaling complex of bacterial chemotaxis (Bass and Falke, 1998; Mehan et al., 2003; Miller et al., 2006). This complex transduces attractant binding into a transmembrane signal that regulates the activity of a cytoplasmic histidine kinase (Baker et al., 2006;...