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Globins and Other Nitric Oxide-Reactive Proteins, Part B, 437 : Volume 437 - Robert Poole

Globins and Other Nitric Oxide-Reactive Proteins, Part B, 437

Volume 437

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Published: 22nd April 2008
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The critically acclaimed laboratory standard for more than forty years, Methods in Enzymology is one of the most highly respected publications in the field of biochemistry. Since 1955, each volume has been eagerly awaited, frequently consulted, and praised by researchers and reviewers alike. Now with over 400 volumes (all of them still in print), the series contains much material still relevant today-truly an essential publication for researchers in all fields of life sciences.

Methods in Enzymology is now available online at ScienceDirect - full-text online of volumes 1 onwards. For more information about the Elsevier Book Series on ScienceDirect Program, please visit:
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This volume is the second of two planned volumes on the topic of globin and other nitric oxide-reactive proteins.

Contributorsp. xvii
Prefacep. xxvii
Volumes in Seriesp. xxix
Nitric Oxide-Metabolising and Detoxifying Enzymesp. 1
Structural Studies on Flavodiiron Proteinsp. 3
Introductionp. 4
Crystallization of Flavodiiron Proteinsp. 4
Diffraction Data Collection, Structure Determination, and Refinementp. 7
Overall Description of Structuresp. 8
Conclusionp. 16
Referencesp. 17
Biochemical, Spectroscopic, and Thermodynamic Properties of Flavodiiron Proteinsp. 21
Introductionp. 22
Cloning of Genes Encoding Flavodiiron Proteins and Their Truncated Domainsp. 24
Production and Purification of Recombinant Flavodiiron Proteinsp. 25
Biochemical Characterization of Flavodiiron Proteinsp. 26
Spectroscopic Propertiesp. 29
Redox Propertiesp. 32
Conclusionsp. 37
Acknowledgmentsp. 42
Referencesp. 42
Kinetic Characterization of the Escherichia coli Nitric Oxide Reductase Flavorubredoxinp. 47
Introductionp. 48
Amperometric Measurementsp. 49
Spectroscopic Measurementsp. 51
Conclusionsp. 61
Acknowledgmentsp. 61
Referencesp. 61
Escherichia coli Cytochrome c Nitrite Reductase NrfAp. 63
Introductionp. 64
Measurement of Cytochrome c Nitrite Reductase-Dependent Consumption of Nitric Oxide in Whole Cellsp. 66
Growth of E. coli Optimized for Cytochrome c Nitrite Reductase Production for Use in Enzyme Purificationp. 66
Purification of Cytochrome c Nitrite Reductasep. 68
Assaying the Cytochrome c Nitrite Reductasep. 69
Crystallization of E. coli Cytochrome c Nitrite Reductasep. 73
Concluding Remarksp. 74
Acknowledgmentsp. 76
Referencesp. 76
The Respiratory Nitric Oxide Reductase (NorBC) from Paracoccus denitrificansp. 79
Introductionp. 80
Purification of Native NorBC from Paracoccus denitrificansp. 82
Purification of Recombinant NorBCp. 85
Amperometric Assays of NO Consumptionp. 86
Pseudoazurin as an Electron Donor in Assays of NorBCp. 88
Preparation of NOR for Spectroscopic Investigationp. 91
Electron Paramagnetic Resonance Spectroscopyp. 96
Concluding Remarksp. 98
Acknowledgmentsp. 99
Referencesp. 99
Redox-Controlled Dinitrosyl Formation at the Diiron-Oxo Center of NorAp. 103
Introductionp. 104
Genetic Context and Expression of the NorA Gene in R. eutrophap. 105
Purification of NorAp. 106
Disulfide Bridges in NorAp. 107
Iron Analysis and Preparation of Apo-NorAp. 108
Interconversion of Redox Forms of NorAp. 109
Generation of NorA-DNIC In Vitrop. 111
Preparation of NorA-DNIC Formed In Vivop. 113
Quantification of NO from NorA-DNICp. 113
Outlookp. 114
Referencesp. 114
Purification and Functional Analysis of Fungal Nitric Oxide Reductase Cytochrome P450norp. 117
Introductionp. 118
Screening of P450nor Activityp. 119
Gas Analysisp. 119
Purification of P450norp. 120
Nitric Oxide Reductase Activity Assayp. 121
Protein Sequencingp. 122
Isolation of cDNAp. 123
Subcellular Fractionation of T. cutaneump. 124
Site-Directed Mutagenesisp. 125
Expression of Recombinant Proteinsp. 126
Purification of Recombinant Proteinsp. 127
Titration of NAD Analogsp. 128
Stopped-Flow Rapid Scan Analysisp. 130
Other Analysisp. 131
Conclusionp. 131
Acknowledgmentsp. 131
Referencesp. 131
A Quantitative Approach to Nitric Oxide Inhibition of Terminal Oxidases of the Respiratory Chainp. 135
Introductionp. 136
Evaluation of Current Techniques for Measuring pNO, pO[subscript 2], and K[subscript M] (O[subscript 2])p. 137
Nitric Oxide Donor Compoundsp. 138
Nitric Oxide Kineticsp. 139
Oxygen Kineticsp. 149
Optical Detection of Enzyme Intermediates in the Presence of Oxygen and NOp. 151
Appendicesp. 153
Acknowledgmentsp. 156
Referencesp. 156
Sensor Proteinsp. 161
Cloning, Expression, and Purification of the N-terminal Heme-Binding Domain of Globin-Coupled Sensorsp. 163
Introductionp. 164
Bioinformatic Search of Globin-Coupled Sensorsp. 164
Functional Analysis of Globin-Coupled Sensorsp. 166
Acknowledgmentsp. 171
Referencesp. 171
Oxygen-Sensing Histidine-Protein Kinases: Assays of Ligand Binding and Turnover of Response-Regulator Substratesp. 173
Introductionp. 174
Assaysp. 175
Acknowledgmentsp. 187
Referencesp. 187
Reactions of Nitric Oxide and Oxygen with the Regulator of Fumarate and Nitrate Reduction, a Global Transcriptional Regulator, during Anaerobic Growth of Escherichia colip. 191
Introductionp. 192
Production of 4Fe-FNR Proteinp. 194
Determination of Iron and Acid-Labile Sulfide Content of FNRp. 197
UV-Visible Absorbance Spectra of FNRp. 198
Cluster Reaction with Nitric Oxide and Oxygenp. 198
Purification of 2Fe-FNRp. 204
Detection of Other Reaction Productsp. 204
Conclusionsp. 206
Referencesp. 207
Genome-Wide Identification of Binding Sites for the Nitric Oxide-Sensitive Transcriptional Regulator NsrRp. 211
Introductionp. 212
Strain Constructionp. 214
Reference and Control Samplesp. 216
Culture Conditionsp. 217
Immunoprecipitation of DNA Targets Associated with NsrRp. 218
DNA Labeling, Microarray Hybridization, and Processingp. 219
Visualization and Analysis of DNA Microarray Datap. 220
A New Statistical Methodology for Treatment of Chip-on-Chip Datap. 222
Conclusionsp. 231
Acknowledgmentsp. 231
Referencesp. 231
Characterization of the Nitric Oxide-Reactive Transcriptional Activator NorRp. 235
Introductionp. 236
Measurement of NorR Activity In Vivop. 237
Measurement of Transcriptional Activation by NorR In Vitrop. 238
Detection of the Ferrous-Nitrosyl Form of NorR by In Vivo Electron Paramagnetic Resonance (EPR)p. 240
In Vitro Reconstitution of the Iron Center in NorRp. 242
Measurement of NO Affinityp. 243
Standardization of the NO Electrodep. 246
Determination of NorRFe(NO) K[subscript d]p. 247
Conclusionsp. 248
Acknowledgmentp. 248
Referencesp. 249
Advanced Spectroscopic Methodsp. 253
Hemoglobins from Mycobacterium tuberculosis and Campylobacter jejuni: A Comparative Study with Resonance Raman Spectroscopyp. 255
Hemoglobin Superfamily: An Overviewp. 256
Microbial Hemoglobinsp. 257
Resonance Raman Spectroscopy: Applications in Hemeproteinsp. 258
Structures and Functions of Microbial Hemoglobinsp. 266
Closing Remarksp. 281
Acknowledgmentsp. 282
Referencesp. 282
The Power of Using Continuous-Wave and Pulsed Electron Paramagnetic Resonance Methods for the Structure Analysis of Ferric Forms and Nitric Oxide-Ligated Ferrous Forms of Globinsp. 287
Introductionp. 288
Electron Paramagnetic Resonance in a Nutshellp. 289
EPR Studies of NO-Ligated Globinsp. 295
EPR Studies of Ferric globinsp. 301
Spin-Labeling Heme Proteinsp. 304
Future Challenges and Possibilitiesp. 305
Acknowledgmentsp. 305
Referencesp. 306
Oxygen Binding to Heme Proteins in Solution, Encapsulated in Silica Gels, and in the Crystalline Statep. 311
Oxygen-Binding Curves to Heme Proteinsp. 313
Determination of OBCs for Hemoglobin in Solutionp. 316
Determination of K[subscript 1] for Hemoglobin in Solution in the Absence of Allosteric Effectorsp. 318
Determination of OBCs for T State Hemoglobin Gels in the Absence and Presence of Allosteric Effectorsp. 318
Determination of OBCs for T State Hemoglobin Crystalsp. 320
Determination of OBCs for Hemocyanin in Solution and in Silica Gelsp. 323
Acknowledgmentsp. 325
Referencesp. 325
Characterization of Ligand Migration Mechanisms inside Hemoglobins from the Analysis of Geminate Rebinding Kineticsp. 329
Introductionp. 330
Principles of Nanosecond Laser Flash Photolysisp. 330
Basic Experimental Layoutsp. 331
Encapsulation of Hbs in Silica Gelsp. 335
Enhancement of Geminate Rebinding and Advantages of Gel Encapsulationp. 336
Extraction of Kinetic Informationp. 337
Acknowledgmentsp. 342
Referencesp. 342
Ligand Dynamics in Heme Proteins Observed by Fourier Transform Infrared Spectroscopy at Cryogenic Temperaturesp. 347
Introductionp. 348
Materialsp. 349
Fourier Transform Infrared Cryospectroscopyp. 353
Low-Temperature FTIR Spectroscopy on NO-Ligated Heme Proteinsp. 365
Concluding Remarksp. 373
Acknowledgmentsp. 374
Referencesp. 374
Time-Resolved X-Ray Crystallography of Heme Proteinsp. 379
Introductionp. 379
Experimentp. 381
Data Processing and Analysisp. 385
A Case Study: Scapharca Dimeric Hemoglobinp. 388
Conclusionsp. 391
Acknowledgmentsp. 393
Referencesp. 393
Structural Dynamics of Myoglobinp. 397
Backgroundp. 398
Crystallographic Studies of Myoglobin Statesp. 399
Experimental Approachesp. 400
Acknowledgmentsp. 413
Referencesp. 413
Use of the Conjugate Peak Refinement Algorithm for Identification of Ligand-Binding Pathways in Globinsp. 417
Introductionp. 418
Exploration of Oxygen-Binding Pathways in Myoglobinp. 418
Theoretical Modelsp. 419
Potential Energy Functionp. 420
Transition Pathwaysp. 421
Methodsp. 425
Resultsp. 429
Conclusionsp. 432
Referencesp. 433
Finding Gas Migration Pathways in Proteins Using Implicit Ligand Samplingp. 439
Introductionp. 440
Methodsp. 442
Example Calculation: Truncated Hemoglobin (trHb) from Paramecium caudatump. 446
Discussionp. 449
Acknowledgmentsp. 455
Referencesp. 456
Identification of Ligand-Binding Pathways in Truncated Hemoglobins Using Locally Enhanced Sampling Molecular Dynamicsp. 459
Introductionp. 460
Molecular Dynamicsp. 462
Locally Enhanced Sampling Molecular Dynamicsp. 465
Methodsp. 466
Resultsp. 468
Conclusionsp. 471
Referencesp. 472
Nitric Oxide Reactivity with Globins as Investigated Through Computer Simulationp. 477
Introductionp. 478
Molecular Dynamics (MD) Methodsp. 479
Quantum Mechanical-Molecular Mechanical Methodsp. 485
Illustrative Examplesp. 488
Ligand Migration Profiles from MSMD and PELE Simulations: Exploring Ligand Entry Pathways in M. tuberculosis trHbNp. 490
Conclusionsp. 494
Acknowledgmentsp. 495
Referencesp. 495
Microbial Responses to Nitric Oxide and Nitrosative Stress: Growth, "Omic," and Physiological Methodsp. 499
Introductionp. 500
Methodsp. 504
Nitric Oxide, NO-Releasing Agents, and Nitrosating Agentsp. 512
Illustrative Results from Applications of These Methodsp. 514
Referencesp. 516
Analysis of Nitric Oxide-Dependent Antimicrobial Actions in Macrophages and Micep. 521
NO.-Dependent Antimicrobial Actions of Murine Macrophagesp. 522
NO.-Dependent Antimicrobial Actions of Human Macrophagesp. 528
NO.-dependent Antimicrobial Actions in Laboratory Micep. 532
Referencesp. 536
Measuring Nitric Oxide Metabolism in the Pathogen Neisseria meningitidisp. 539
Introductionp. 540
Safety Aspects of Handling N. meningitidis in the Laboratoryp. 541
Metabolism of Neisseria sp.p. 541
Experimental Approaches to Analyzing Nitrogen Metabolism Relevant to NOp. 544
Simultaneous Measurement of Oxygen and NO during Pure Culture of N. meningitidisp. 547
Measurement of NO Production/Disappearance in Tissue Culture Using Human Monocyte-Derived Macrophagesp. 555
Referencesp. 558
Localization of S-Nitrosothiols and Assay of Nitric Oxide Synthase and S-Nitrosoglutathione Reductase Activity in Plantsp. 561
Introductionp. 562
Determination of L-Arginine-Dependent NOS Activity by Ozone Chemiluminescence in Plant Tissuesp. 563
Assay of GSNOR Activityp. 566
Localization of S-Nitrosothiols and S-Nitrosoglutathione in Plant Tissues by Confocal Laser-Scanning Microscopy (CLSM)p. 567
Conclusionp. 571
Acknowledgmentsp. 572
Referencesp. 572
Methods for Nitric Oxide Detection during Plant-Pathogen Interactionsp. 575
Introductionp. 576
Nitric Oxide Detection by Mass Spectrometryp. 577
Nitric Oxide Detection by Laser Photoacoustic Spectroscopyp. 579
Nitric Oxide Detection by Chemiluminescencep. 582
Nitric Oxide Detection by Hemoglobin Conversionp. 583
Nitric Oxide Detection by Electron Paramagnetic Resonance (EPR) Spin Trapp. 585
Nitric Oxide Detection Using Diaminofluoresceinsp. 587
Conclusionp. 590
Referencesp. 591
Bioimaging Techniques for Subcellular Localization of Plant Hemoglobins and Measurement of Hemoglobin-Dependent Nitric Oxide Scavenging In Plantap. 595
Introductionp. 596
Measuring Hemoglobin-Dependent NO Scavengingp. 596
Techniques for Determination of Subcellular Localization of Plant Hemoglobinsp. 597
Imaging of Hemoglobin-Dependent NO Scavenging in Arabidopsis Plantsp. 598
Engineering of GLB1-GFP/GLB2-GFP Constructs and Microscopic Analysis of A. thaliana Plants Expressing GFP-Tagged Hemoglobinp. 600
Referencesp. 603
Use of Recombinant Iron-Superoxide Dismutase as A Marker of Nitrative Stressp. 605
Introductionp. 606
Immunodetection of Nitrated Proteins: Metal-Mediated Tyrosine Nitration of BSAp. 608
Tyrosine Nitration of Purified Recombinant Vu_FeSOD Affects its Enzymatic Activityp. 610
Tyrosine Nitration in Vu_FeSOD can be Estimated Using Antibodies Against 3-Nitrotyrosinep. 612
SIN-1-Dependent Vu_FeSOD Nitration can be Detected by the Loss of Enzymatic Activityp. 612
Acknowledgmentsp. 616
Referencesp. 616
Author Indexp. 619
Subject Indexp. 647
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ISBN: 9780123742780
ISBN-10: 0123742781
Series: Methods in Enzymology : Book 437
Audience: Professional
Format: Hardcover
Language: English
Number Of Pages: 712
Published: 22nd April 2008
Publisher: Elsevier Science Publishing Co Inc
Country of Publication: US
Dimensions (cm): 22.9 x 15.2  x 3.3
Weight (kg): 1.09
Edition Number: 1