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." This volume, the third in a three-volume treatment edited by the same group of editors, includes a wide range of methods, including those dealing with the Sln-1 kinase pathway, Triazole sensitivity in C. albicans, and Histidine kinases in cyanobacteria circadian clock. * Includes time-tested core methods and new innovations applicable to any researcher studing two-component signaling systems or histidine kinases * Methods included are useful to both established researchers and newcomers to the field * Relevant background and reference information given for procedures can be used as a guide to developing protocols in a number of disciplines
Author(s): Melvin I. Simon, Brian Crane, Alexandrine Crane
Series: Methods in Enzymology 471
Edition: 1
Publisher: Elsevier, Academic Press
Year: 2010
Language: English
Pages: 496
Cover......Page 1
Methods in Enzymology, Volume 471......Page 2
Series Editors......Page 3
ISBN 978-0-12-381347-3......Page 4
Contributors......Page 5
Contents of Previous Volumes......Page 11
Abstract......Page 39
Overview......Page 40
Sources of Cross-Talk......Page 41
Cross-Talk Suppression......Page 42
Transcriptional Reporters......Page 44
Response Regulator Localization......Page 46
Phosphatase Cross-Talk......Page 51
Concluding Remarks......Page 52
References......Page 53
Abstract......Page 55
Introduction......Page 56
Operon database......Page 62
Expression and purification of recombinant human NDPK, standardized activity assays......Page 411
Comparison to known operons......Page 63
Single protein architecture filtering......Page 64
Final dataset......Page 65
DCA: Direct Coupling Analysis......Page 66
Weighting......Page 67
Frequency counts......Page 68
Global statistical modeling......Page 69
Residue selection......Page 70
Initialization......Page 71
Belief Propagation......Page 72
Susceptibility Propagation......Page 73
Does the model describe empirical data within threshold?......Page 74
Direct information......Page 75
Nonstandard Linear Algebra Functions......Page 76
References......Page 77
References......Page 454
Abstract......Page 80
Introduction......Page 81
Structure-based simulations......Page 84
Deriving parameters for molecular docking simulations......Page 86
Test: Docking of the Spo0B/Spo0F complex......Page 87
Summary......Page 91
References......Page 92
Abstract......Page 96
Introduction......Page 97
Protein purification and phosphorylation......Page 99
The RQF instrument......Page 100
Experimental design......Page 101
Rapid quench flow experiments......Page 104
Data analysis and interpretation......Page 106
Conclusion......Page 110
References......Page 111
Abstract......Page 113
Introduction......Page 114
Comparison Between the Cytoplasmic Domains of E. coli and Typhi EnvZ Proteins......Page 116
Purification of the Recombinant MBP Proteins by FPLC......Page 118
Results......Page 174
Results......Page 120
References......Page 121
Measurement of Response Regulator Autodephosphorylation Rates Spanning Six Orders of Magnitude......Page 124
Abstract......Page 125
Introduction......Page 160
Purification of Response Regulator Proteins......Page 126
General Considerations for Autodephosphorylation Assays......Page 127
Assay of Autodephosphorylation by Loss of 32P......Page 129
Structural Analysis of KinC......Page 130
Procedure for autodephosphorylation assay of E. coli [32P]CheY or B. subtilis [32P]Spo0F response regulators.........Page 131
Modification for phosphatase activity or slow phosphotransfer......Page 133
Modification for fast autodephosphorylation......Page 134
Modification for small molecule phosphodonors......Page 135
Gene Disruption......Page 179
Kinetic theory......Page 136
Fluorescence/pH jump method......Page 137
Assay of Autodephosphorylation by Pi Release......Page 141
Synthesis of phosphoramidate and monophosphoimidazole......Page 142
Assay of Autodephosphorylation from Systems of Reactions......Page 143
Statement of the problem......Page 182
In Vitro Biochemical Analysis of TCS Proteins......Page 302
Future Prospects......Page 145
References......Page 146
Abstract......Page 150
Introduction......Page 151
Design of Chimeras of HAMP Domains Fused to the AC Rv3645 Catalytic Domain......Page 152
Choice of Vector and Cloning Strategy......Page 153
Filter-Based Assay of Alkali-Stable, Acid-Labile Protein Phosphorylation (Nytran Assay)......Page 445
Example of Applications of the Method and Results......Page 155
Phosphotransfer......Page 156
References......Page 157
Abstract......Page 159
Cloning and purification of LOV-HKs......Page 161
Assesment of LOV-kinase purity......Page 163
Kinase photochemical-activity assay......Page 164
Safe-light conditions for sample manipulation......Page 165
Overview......Page 223
Concluding Remarks and Future Perspectives......Page 166
Acknowledgments......Page 167
Characterization of Bacteriophytochromes from Photosynthetic Bacteria......Page 169
The Arabidopsis Circadian Clock......Page 170
Cloning BphP in Expression Vector......Page 173
Autophosphorylation......Page 175
Gel mobility shift assay......Page 177
Synthesis of [32P] labeled dilithium acetyl phosphate......Page 181
Derivation of the unknown parameters from steady-state spectra......Page 184
Absolute quantum yields......Page 188
References......Page 192
Biophysical Assays for Protein Interactions in the Wsp Sensory System and Biofilm Formation......Page 194
Introduction......Page 195
Materials for the E. coli system......Page 328
Size-exclusion chromatography......Page 196
Analytical ultracentrifugation......Page 197
SEC-coupled static multiangle light scattering......Page 198
Transformation of M. xanthus by electroporation......Page 200
Detection of Cysteine S-Thiolation in Tdh1 GAPDH......Page 203
Experimental Considerations......Page 204
Case Studies......Page 207
Phosphorylation and dephosphorylation of ATP-citrate lyase......Page 416
WspR......Page 209
Concluding Remarks......Page 213
References......Page 214
High-Throughput Screening of Bacterial Protein Localization......Page 218
Abstract......Page 219
Introduction......Page 312
Pipeline Overview......Page 220
Construction of a Caulobacter ORFeome......Page 221
PRR protein detection: immunoblots......Page 222
Measurement of PHPT-1 activity......Page 374
Designing xylose-inducible mCherry fusion destination vectors......Page 224
Procedure for the in vivo LR reaction......Page 225
Results......Page 227
Cotyledon movement analysis......Page 228
Overview......Page 230
Protocol for image scoring and validation......Page 231
Results......Page 232
Conclusion......Page 234
Acknowledgments......Page 235
References......Page 236
In Vitro and In Vivo Analysis of the ArcB/A Redox Signaling Pathway......Page 238
Dissection of the Arc components to individual subdomains......Page 241
ArcB-enriched inverted vesicles......Page 242
Expression and purification of soluble forms of full-length ArcB or its subdomains......Page 244
Autophosphorylation and transphosphorylation assays......Page 245
Transphosphorylation......Page 246
Preparation of Radioactive Phospho-HPt Factor, and In Vitro Assay of Phosphotransfer to RR......Page 375
RR-P dephosphorylation......Page 247
Discussion......Page 248
Phosphatase assays......Page 249
Redox regulation of ArcB autophosphorylation......Page 250
Blocking cysteine residues with S-methyl methanethiosulfonate......Page 251
Tagging Cys residues with methoxy-poly(ethylene glycol) maleimide......Page 252
In Vivo Characterization of the Arc TCS......Page 253
Construction of a deletion mutant and chromosomal gene replacement......Page 254
Detection of signaling defects in the Arc TCS......Page 255
In vivo reconstitution of the Arc signaling pathways......Page 256
Ablations and transplantations......Page 257
References......Page 259
Potassium Sensing Histidine Kinase in Bacillus subtilis......Page 262
Introduction......Page 263
Expression and purification of NDPK and PHPT-1......Page 266
Quantitative Analysis of the Activation of KinC......Page 269
Monitoring the Signals Using Indirect Measurements......Page 277
Applications of the System Signal-Kinase......Page 278
Conclusions......Page 281
References......Page 307
Two-Component Systems and Regulation of Developmental Progression in Myxococcus xanthus......Page 285
Introduction......Page 286
Generation of In-Frame Deletions or Point Mutations in the M. xanthus Genome......Page 289
Plasmid construction......Page 290
Preparation of electrocompetent M. xanthus......Page 293
Analysis of development on nutrient limited agar plates......Page 295
Analysis of development in submerged culture......Page 296
Analysis of sporulation efficiency......Page 297
Cell population analysis......Page 298
Overview and general considerations......Page 299
RT-PCR analysis of developmental markers......Page 300
Western blot analysis of developmental markers......Page 301
Protein overproduction and purification......Page 303
Autophosphorylation of histidine kinases......Page 304
Production of acetyl-[32P]......Page 305
Phosphotransfer analysis......Page 306
Two-Component Signaling to the Stress MAP Kinase Cascade in Fission Yeast......Page 311
Affinity purification of tagged proteins from S. pombe cell lysate......Page 314
Mcs4 RR-MAPKKK interactions......Page 315
Interaction between the Mpr1 HPt protein and Mcs4 RR......Page 317
Interaction of Tdh1 GAPDH with Mcs4 RR and MAPKKKs......Page 318
References......Page 320
Genetic and Biochemical Analysis of the SLN1 Pathway in Saccharomyces cerevisiae......Page 322
Introduction......Page 323
Activation of the SLN1 pathway with zymolyase......Page 331
Northern analysis of SLN1-dependent genes......Page 332
Osmotic activation of the HOG1 pathway......Page 333
GST-Sln1-HK and GST-Sln1-HK-Rec domains......Page 334
Sln1, Ssk1, and Skn7 receiver domains......Page 336
Ypd1 phosphorelay protein......Page 338
Autophosphorylation of GST-tagged Sln1-HK bound to glutathione-coupled resin......Page 339
Regulation of ACL activity by PHPT-1......Page 340
Phosphorylation of Ssk1 and Skn7 receiver domains for half-life studies, mutant studies, or phosphotransfer assays
......Page 342
Measurement of the phosphorylated lifetime of SLN1 pathway components......Page 343
Phosphotransfer or phosphorelay experiments......Page 345
References......Page 346
Analysis of Mitogen-Activated Protein Kinase Phosphorylation in Response to Stimulation of Histidine Kinase Signaling Pathways in Neurospora
......Page 349
Introduction......Page 350
Reagents......Page 352
Growth and collection of cultures......Page 353
Reagents......Page 354
Preparation of protein extracts......Page 357
SDS-PAGE electrophoresis and transfer of protein to membrane......Page 358
Western blot analysis using MAPK antibodies......Page 359
Results......Page 360
Adapting the MAPK Assay......Page 361
References......Page 405
References......Page 362
Biochemical Characterization of Plant Hormone Cytokinin-Receptor Histidine Kinases Using Microorganisms......Page 365
Introduction......Page 366
Overview and logic behind......Page 370
Semi-quantitative protocol......Page 373
Overview and logic behind......Page 377
Materials for the yeast system......Page 378
Protocol of Histidine Kinase Assay in S. cerevisiae......Page 379
Protocol of Cytokinin-Binding Assay by Using S. pombe Membranes Enriched in AHK4/CRE1......Page 380
References......Page 381
Abstract......Page 387
Immunodetection of GFP-tagged PRR proteins from Arabidopsis extracts......Page 390
Protein extract preparation for PRR family members......Page 391
Immunoprecipitation/coimmunoprecipitation......Page 392
Determination of phosphorylation states of PRR family members......Page 393
Localization of GFP-tagged PRR proteins in leek cells......Page 394
Protocol: transient expression of 35S:GFP:PRR clones in leek cells......Page 395
Immunolocalization of GFP-tagged PRR proteins in Arabidopsis plants......Page 396
Exploring the Circadian Phenotypes of prr Mutants......Page 397
Generating versatile LUCIFERASE (LUC) fusion constructs......Page 399
Transient rhythmic assay in Nicotiana benthamiana......Page 401
Luciferase assays on a Topcount......Page 403
Abstract......Page 409
Introduction......Page 410
Assay for NDPK activity......Page 412
Expression and purification of human recombinant PHPT-1......Page 413
Phosphorylation and dephosphorylation of Gbeta......Page 414
Generation and amplification of recombinant adenoviruses......Page 417
Adenoviral overexpression of WT or phosphorylation-deficient Gbeta1gamma2-dimers in cardiomyocytes......Page 419
Determination of basal cAMP synthesis in NRCM......Page 421
Preparation of cell extracts and analysis of PHPT-1 expression and ACL activity......Page 422
Regulation of KCa3.1 activity by NDPKB and PHPT-1......Page 423
Culture and transfection of HEK293 cells......Page 425
Recording of whole-cell currents......Page 428
Single-channel recording......Page 429
References......Page 430
Abstract......Page 433
Introduction......Page 434
Chemical Phosphorylation of Histone H4 Proteins and Peptides......Page 435
Detection of Phosphohistidine-Phosphoamino Acid Analysis......Page 436
In-Gel Kinase Assay......Page 446
Phosphorylation and Thiophosphorylation Site Analysis by Edman Sequencing......Page 449
Mass Spectrometric Phosphopeptide Analysis......Page 451
Author Index......Page 457
Subject Index......Page 465
