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Contents :
1. Tyrosine Fluorescence and Phosphorescence from Proteins and
Polypeptides
J. B. Alexander Ross, William R. Laws, Kenneth W. Rousslang, and
Herman R. Wyssbrod
1.1. Historical Perspective and Background. . . . . . . . . . . . . . . 1
1.2. The Absorption Properties of Tyrosine. . . . . . . . . . . . . . . 2
1.3. The Excited Singlet and Triplet States of Tyrosine and
Tyrosinate . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
1.3.1. The Zero-Field Splittings of the Triplet State. . . . . . . 5
1.3.2. Excited-State Decay Kinetics. . . . . . . . . . . . . . . . 7
1.4. Quenching Mechanisms of Tyrosine Emission in Polypeptides
and Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . .12
1.4.1. The Peptide Bond. . . . . . . . . . . . . . . . . . . . . .12
1.4.2. Singlet-Singlet and Triplet-Triplet Resonance Energy
Transfer. . . . . . . . . . . . . . . . . . . . . . . . . .13
1.4.3. Disulfide Bonds and Sulfhydryl Groups . . . . . . . . . . .17
1.4.4. Interactions with Ionizable Side Chains and Proton
Acceptors . . . . . . . . . . . . . . . . . . . . . . . . .20
1.5. Emission from Polypeptides and Proteins. . . . . . . . . . . . . .21
1.5.1. Fluorescence of Tyrosine. . . . . . . . . . . . . . . . . .22
1.5.2. Fluorescence of Tyrosinate. . . . . . . . . . . . . . . . .43
1.5.3. Phosphorescence and ODMR of Proteins and Polypeptides . . .50
1.6. Tyrosine as an Excited-State Probe for Conformation and
Dynamics . . . . . . . . . . . . . . . . . . . . . . . . . . . . .52
References . . . . . . . . . . . . . . . . . . . . . . . . . . . .53
2. Fluorescence and Dynamics in Proteins
A. P. Demchenko
2.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . .65
2.2. Dynamics in Proteins . . . . . . . . . . . . . . . . . . . . . . .68
2.2.1. Structural Hierarchy and Degrees of Mobility. . . . . . . .68
2.2.2. Distribution of Microstates . . . . . . . . . . . . . . . .70
2.2.3. Analysis of Motions Using Time-Resolved Methods . . . . . .71
2.3. Decay and Quenching of Fluorescence. . . . . . . . . . . . . . . .74
2.3.1. Emission Decay Kinetics . . . . . . . . . . . . . . . . . .74
2.3.2. Fluorescence Quenching by Intrinsic Quenchers . . . . . . .77
2.3.3. Fluorescence Quenching by Extrinsic Quenchers . . . . . . .78
2.4. Rotation of Aromatic Groups. . . . . . . . . . . . . . . . . . . .81
2.4.1. Fluorescence Polarization Studies with and without Time
Resolution. . . . . . . . . . . . . . . . . . . . . . . . .81
2.4.2. Models of Rotations . . . . . . . . . . . . . . . . . . . .83
2.5. Fluorescence Spectroscopy of Molecular Relaxation. . . . . . . . .85
2.5.1. Dynamic Reorientation of Dipoles in the Fluorophore
Environment . . . . . . . . . . . . . . . . . . . . . . . .85
2.5.2. The Two-State Model of Relaxation . . . . . . . . . . . . .87
2.5.3. Continuous Model of Relaxation. . . . . . . . . . . . . . .88
2.5.4. Site-Photoselection Model . . . . . . . . . . . . . . . . .91
2.6. Molecular Relaxation and Dynamics of Dipoles in the Protein
Globule. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .95
2.6.1. Relaxational Shift of Steady-State Spectra. . . . . . . . .95
2.6.2. Time-Resolved Spectra . . . . . . . . . . . . . . . . . . .96
2.6.3. Red-Edge Excitation Spectroscopy. . . . . . . . . . . . . .97
2.7. Conclusion and Future Prospects. . . . . . . . . . . . . . . . . 104
References . . . . . . . . . . . . . . . . . . . . . . . . . . . 106
3. Tryptophan Phosphorescence from Proteins at Room Temperature
Jane M. Vanderkooi
3.1. Background . . . . . . . . . . . . . . . . . . . . . . . . . . . 113
3.2. Triplet State Formation and Disappearance. . . . . . . . . . . . 114
3.2.1. Energy Diagram. . . . . . . . . . . . . . . . . . . . . . 114
3.2.2. General Considerations of Phosphorescence Yield . . . . . 115
3.2.3. Measurement of Phosphorescence. . . . . . . . . . . . . . 116
3.3. Tryptophan Phosphorescence Emission from Proteins. . . . . . . . 117
3.3.1. Comparison of Fluorescence and Phosphorescence
Emission Spectra. . . . . . . . . . . . . . . . . . . . . 117
3.3.2. Delayed Fluorescence. . . . . . . . . . . . . . . . . . . 118
3.3.3. Lifetime of Tryptophan Phosphorescence in Proteins. . . . 119
3.3.4. What Affects the Phosphorescence Lifetime?. . . . . . . . 121
3.3.5. Phosphorescence Quenching by External Molecules . . . . . 123
3.3.6. Phosphorescence Lifetimes to Measure Conformational
Changes in Proteins . . . . . . . . . . . . . . . . . . . 128
3.4. Phosphorescence Anisotropy and Rotational Motion . . . . . . . . 130
3.4.1. Phosphorescence Anisotropy. . . . . . . . . . . . . . . . 130
3.4.2. Anisotropy to Study Proteins. . . . . . . . . . . . . . . 131
3.5. Tryptophan Phosphorescence from Cells. . . . . . . . . . . . . . 131
3.6. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . 132
References . . . . . . . . . . . . . . . . . . . . . . . . . . . 132
4. Fluorescence Studies of Nucleic Acids: Dynamics, Rigidities, and
Structures
J. Michael Schurr, Bryant S. Fujimoto, Pengguang Wu, and Lu Song
4.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . 137
4.2. Rotational Dynamics of DNA . . . . . . . . . . . . . . . . . . . 138
4.2.1. Background. . . . . . . . . . . . . . . . . . . . . . . . 138
4.2.2. Pertinent Questions and Problems. . . . . . . . . . . . . 140
4.2.3. Theory. . . . . . . . . . . . . . . . . . . . . . . . . . 145
4.2.4. Instrumentation . . . . . . . . . . . . . . . . . . . . . 169
4.2.5. Protocol and Data Analysis. . . . . . . . . . . . . . . . 170
4.2.6. Experimental Results. . . . . . . . . . . . . . . . . . . 172
4.3. Rotational Dynamics of DNA in Nucleosomes, Chromatin,
Viruses, and Sperm . . . . . . . . . . . . . . . . . . . . . . . 211
4.3.1. Nucleosomes . . . . . . . . . . . . . . . . . . . . . . . 211
4.3.2. Chromatin . . . . . . . . . . . . . . . . . . . . . . . . 213
4.3.3. Viruses . . . . . . . . . . . . . . . . . . . . . . . . . 214
4.3.4. Sperm . . . . . . . . . . . . . . . . . . . . . . . . . . 214
4.4. Steady-State Studies of DNA Dynamics . . . . . . . . . . . . . . 215
4.5. DNA Dynamics by Fluorescence Microscopy. . . . . . . . . . . . . 216
4.6. Dynamics of tRNAs. . . . . . . . . . . . . . . . . . . . . . . . 218
4.6.1. Ethidium Fluorescence . . . . . . . . . . . . . . . . . . 218
4.6.2. Wyebutine Fluorescence. . . . . . . . . . . . . . . . . . 220
4.7. Summary and Outlook. . . . . . . . . . . . . . . . . . . . . . . 222
References . . . . . . . . . . . . . . . . . . . . . . . . . . . 222
5. Fluorescence in Membranes
Christopher D. Stubbs and Brian Wesley Williams
5.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . 231
5.2. Fluorescence Lifetimes . . . . . . . . . . . . . . . . . . . . . 232
5.2.1. The Use of Fluorescence Lifetimes for Membrane
Organizational Studies. . . . . . . . . . . . . . . . . . 232
5.2.2. Fluorescence Lifetime Distributions . . . . . . . . . . . 233
5.2.3. Excimer Probes. . . . . . . . . . . . . . . . . . . . . . 239
5.3. Fluorescence Anisotropy. . . . . . . . . . . . . . . . . . . . . 239
5.3.1. Anisotropy Parameters . . . . . . . . . . . . . . . . . . 240
5.3.2. Time-Resolved Anisotropy. . . . . . . . . . . . . . . . . 241
5.3.3. Applications to Membrane Studies. . . . . . . . . . . . . 245
5.3.4. Fluorescent Probes for Lifetime and Anisotropy Studies. . 246
5.4. Fluorescence Energy Transfer . . . . . . . . . . . . . . . . . . 248
5.4.1. Surface Distribution of Fluorophore-Labeled Lipids. . . . 249
5.4.2. Location of the Longitudinal and Lateral Position of
Membrane Proteins . . . . . . . . . . . . . . . . . . . . 251
5.4.3. Protein-Protein Associations. . . . . . . . . . . . . . . 252
5.5. Fluorescence Quenching . . . . . . . . . . . . . . . . . . . . . 252
5.5.1. Determination of Partitioning and Binding of Fluorophore
Quenchers to Membranes. . . . . . . . . . . . . . . . . . 253
5.5.2. Location of Fluorophores. . . . . . . . . . . . . . . . . 257
5.6. Solvent Relaxation . . . . . . . . . . . . . . . . . . . . . . . 257
5.7. Surface Charge . . . . . . . . . . . . . . . . . . . . . . . . . 259
5.8. Future Directions. . . . . . . . . . . . . . . . . . . . . . . . 262
References . . . . . . . . . . . . . . . . . . . . . . . . . . . 263
6. Fluorescence and Immunodiagnostic Methods
Thomas M. Li and Richard F. Parrish
6.1. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . 273
6.2. Assay Formats . . . . . . . . . . . . . . . . . . . . . . . . . 274
6.3. Fluorescence Polarization Immunoassay . . . . . . . . . . . . . 274
6.4. Substrate-Labeled Fluorescent Immunoassay . . . . . . . . . . . 276
6.5. Intra-Molecularly Quenched Fluorescent Immunoassay. . . . . . . 278
6.6. Homogeneous Fluorescent Immunoassay in a Dry Reagent
Format. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281
6.7. Fluorescence Excitation Transfer Immunoassay. . . . . . . . . . 281
6.8. Design of Fluorescent Probes. . . . . . . . . . . . . . . . . . 282
6.9. Phycobiliproteins . . . . . . . . . . . . . . . . . . . . . . . 284
6.10. Phase-Resolved Fluorescence Immunoassay . . . . . . . . . . . . 285
6.11. Time-Resolved Fluorescence Immunoassay. . . . . . . . . . . . . 286
6.12. Conclusion. . . . . . . . . . . . . . . . . . . . . . . . . . . 286
References. . . . . . . . . . . . . . . . . . . . . . . . . . . 287
7. Total Internal Reflection Fluorescence
Daniel Axelrod, Edward H. Hellen, and Robert M. Fulbright
7.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . 289
7.2. Theory of TIR Excitation . . . . . . . . . . . . . . . . . . . . 290
7.2.1. Single interface. . . . . . . . . . . . . . . . . . . . . 290
7.2.2. Intermediate Layer. . . . . . . . . . . . . . . . . . . . 295
7.3. Emission by Fluorophores near a Surface. . . . . . . . . . . . . 298
7.3.1. Description of the Model. . . . . . . . . . . . . . . . . 299
7.3.2. Mathematical and Physical Basis . . . . . . . . . . . . . 300
7.3.3. Graphical Results . . . . . . . . . . . . . . . . . . . . 304
7.3.4. Theoretical Results for a Distribution of Dipoles:
Random Orientations . . . . . . . . . . . . . . . . . . . 309
7.3.5. Consequences for Experiments. . . . . . . . . . . . . . . 310
7.4. TIRF for a Microscope. . . . . . . . . . . . . . . . . . . . . . 313
7.4.1. Inverted Microscope . . . . . . . . . . . . . . . . . . . 314
7.4.2. Upright Microscope. . . . . . . . . . . . . . . . . . . . 316
7.4.3. Prismless TIRF. . . . . . . . . . . . . . . . . . . . . . 316
7.4.4. TIRF Interference Fringes . . . . . . . . . . . . . . . . 317
7.4.5. General Experimental Suggestions. . . . . . . . . . . . . 319
7.5. Applications of TIRF . . . . . . . . . . . . . . . . . . . . . . 320
7.5.1. Binding of Proteins and Probes to Artificial Surfaces . . 320
7.5.2. Concentration of Molecules near Surfaces. . . . . . . . . 323
7.5.3. Orientation, Rotation, and Fluorescence Lifetime of
Molecules near Surfaces . . . . . . . . . . . . . . . . . 324
7.5.4. Qualitative Observation of Labeled Cells. . . . . . . . . 326
7.5.5 Fluorescence Energy Transfer and TIRF . . . . . . . . . . 329
7.5.6 Reaction Rates at Biosurfaces . . . . . . . . . . . . . . 330
7.5.7. TIRF Combined with Fluorescence Correlation
Spectroscopy (FCS). . . . . . . . . . . . . . . . . . . . 334
7.6. Summary and Comparisons. . . . . . . . . . . . . . . . . . . . . 335
References . . . . . . . . . . . . . . . . . . . . . . . . . . . 337
8. Microparticle Fluorescence and Energy Transfer
L. M. Folan and S. Arnold
8.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . 345
8.1.1. Fluorescence from a Microparticle . . . . . . . . . . . . 345
8.1.2. Nature of the Effects . . . . . . . . . . . . . . . . . . 346
8.2. Excitation Spectroscopy. . . . . . . . . . . . . . . . . . . . . 347
8.2.1. Interaction of a Plane Wave with a Sphere . . . . . . . . 347
8.2.2. Excitation of a Dipole and Photoselection . . . . . . . . 352
8.2.3. Experiments . . . . . . . . . . . . . . . . . . . . . . . 356
8.3. Emission Spectroscopy. . . . . . . . . . . . . . . . . . . . . . 366
8.3.1. Interaction between an Excited Electronic State and a
Microsphere: Radiative and Nonradiative Decay Rates . . . 366
8.3.2. Angular Intensity Distribution. . . . . . . . . . . . . . 370
8.3.3. Energy Transfer . . . . . . . . . . . . . . . . . . . . . 371
8.3.4. Experiments . . . . . . . . . . . . . . . . . . . . . . . 376
8.4. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . 384
References . . . . . . . . . . . . . . . . . . . . . . . . . . . 384
Index. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 387
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