Fluorescence Spectroscopy

Cover Page......Page 1
Series Editors......Page 2
Copyright Page......Page 3
Contributors to Volume 450......Page 4
Preface: Methods in Enzymology (Fluorescence 2008)......Page 8
Volumes in Series......Page 10
A Method in Enzymology for Measuring Hydrolytic Activities in Live Cell Environments......Page 36
Protease Substrate Design......Page 37
Protease Specificity......Page 40
Cell-Permeable Fluorogenic Probes......Page 42
Cellular organization: Ordering of the activation of proteases in live cells......Page 43
Ordering of activation of proteases involved in intracellular protease activation cascades......Page 45
Subcellular localization of protease activities......Page 46
Quantification and visualization of cell-mediated cytotoxicity......Page 47
Measurement of an early event in inflammation: Activation of caspase 1 in macrophages......Page 48
Measurement of migration and invasiveness in metastatic cancer cells......Page 49
Measurement of nuclease activity and hybridization with cell-permeable fluorogenic oligonucleotides inside live cells......Page 50
Conclusions......Page 51
References......Page 52
Heterogeneity of Fluorescence Determined by the Method of Area-Normalized Time-Resolved Emission Spectroscopy......Page 55
Introduction......Page 56
TRES and TRANES Methods......Page 58
TRES and TRANES Spectra of Simple Cases......Page 59
Physical Significance of TRANES and Isoemissive Point......Page 63
TRES and TRANES of Fluorophores in Microheterogeneous Media......Page 64
Fluorescence in Microheterogeneous and Biological Media: Special Cases......Page 66
References......Page 67
Multiparametric Probing of Microenvironment with Solvatochromic Fluorescent Dyes......Page 70
"Universal" and "Specific" Noncovalent Interactions
......Page 72
The Methodology of Multiparametric Approach with Application of 3-Hydroxyflavone Dyes......Page 73
Position of absorption band......Page 76
Stokes shift of the N* and T* bands......Page 77
Ratio of intensities of the N* and T* bands (IN*/IT*)......Page 78
Algorithm for Multiparametric Probing Based on Parameters of Absorption and Dual Emission......Page 80
Probing binary solvent mixtures......Page 82
Probing protein-binding sites......Page 83
H-bonding heterogeneity within the sites of the same polarity......Page 84
Site heterogeneity: Lipid bilayers and protein-labeling sites......Page 85
Applicability of multiparametric approach to other solvatochromic dyes......Page 86
Concluding Remarks......Page 88
References......Page 89
Site-Selective Red-Edge Effects......Page 92
Introduction......Page 93
Molecular Disorder and the Origin of Red-Edge Effects......Page 94
The Principle of Photoselection......Page 95
Ground-State Heterogeneity......Page 98
The Magnitude of Red-Edge Excitation Fluorescence Shift and Its Connection with Dielectric Relaxations......Page 100
Red-Edge Effect with High Resolution in Time......Page 104
Red-Edge Effects in Excited-State Reactions......Page 105
Fluorescent Probes for Optimal Observation of Red-Edge Effects......Page 107
Peculiarities of the Red-Edge Effects of Indole and Tryptophan......Page 108
Conclusions......Page 109
References......Page 110
Fluorescence Approaches to Quantifying Biomolecular Interactions......Page 112
Fluorescence Observables......Page 113
Foumlrster resonance energy transfer......Page 114
Fluorescence correlation spectroscopy......Page 115
Fluorescence anisotropy......Page 117
Choosing fluorescence probes......Page 118
Protein-ligand interactions......Page 119
Protein-nucleic acid interactions......Page 124
Aqueous phase protein-protein interactions......Page 129
Protein-protein interactions in or at the cell membrane......Page 131
References......Page 136
Forster Resonance Energy Transfer Measurements of Transmembrane Helix Dimerization Energetics......Page 140
Challenges in Quantitative Measurements of Interactions Between TM Helices in Bilayers......Page 141
Multilamellar vesicles......Page 143
Surface-supported bilayers......Page 144
FRET Due to Random Colocalization of Donors and Acceptors (Proximity FRET)......Page 147
Direct calculation of FRET efficiencies from donor quenching......Page 148
The EmEx-FRET method......Page 149
EmEx-FRET theory......Page 151
EmEx-FRET protocol......Page 152
Theory and protocol......Page 155
Thermodynamics of protein homodimerization in lipid bilayer membranes: Wild-type FGFR3 TM domain, and the pathogenic Ala391Glu, Gly380Arg, and Gly382Asp mutants......Page 157
References......Page 158
Application of Single-Molecule Spectroscopy in Studying Enzyme Kinetics and Mechanism......Page 161
Introduction......Page 162
Experimental Considerations......Page 166
Sample Preparation......Page 168
Instrumentation......Page 170
Data Analysis of Single-Molecule Trajectories......Page 173
Static heterogeneity......Page 177
Dynamic heterogeneity......Page 178
Kinetics of a single oligomeric enzyme molecule......Page 179
Additional Considerations......Page 183
Summary......Page 186
References......Page 187
Ultrafast Fluorescence Spectroscopy via Upconversion: Applications to Biophysics......Page 190
Introduction......Page 191
Basic Concepts......Page 193
Phase-matching angle......Page 194
Spectral bandwidth......Page 195
Acceptance angle......Page 196
Group velocity mismatch......Page 197
Sample handling......Page 198
Upconversion Spectrophotofluorometer and Experimental Considerations......Page 199
Ultrafast Photophysics of Single Tryptophan, Peptides, Proteins, and Nucleic Acids......Page 202
Solvent relaxation of tryptophan in water......Page 204
QSSQ of Trp in dipeptides......Page 205
Ultrafast fluorescence dynamics of proteins......Page 206
Ultrafast dynamics in DNA......Page 208
References......Page 210
Use of Fluorescence Resonance Energy Transfer (FRET) in Studying Protein-induced DNA Bending......Page 215
Introduction......Page 216
Preparation of Labeled DNA Duplexes......Page 217
Fluorescence Resonance Energy Transfer......Page 219
FRET in Studying Large Protein-Induced DNA Bends......Page 221
FRET in Studying Small Protein-Induced DNA Bends......Page 224
Construction of a U-shaped double bulged fluorophores labeled DNA......Page 225
FRET titration of the U-shaped DNA......Page 227
Acknowledgment......Page 228
References......Page 229
Fluorescent Pteridine Probes for Nucleic Acid Analysis......Page 230
Introduction......Page 231
Intensity and spectral shifts......Page 234
Stability......Page 235
Participation in base pairing......Page 236
Conservation of phosphoramidite......Page 237
Deprotection......Page 238
Characterization of Pteridine-Containing Sequences......Page 239
Using fluorescence intensity changes......Page 240
Fluorescence characterization of A-tracts using 6MAP......Page 241
Temperature-dependent behavior of A-tract duplexes......Page 243
Bulge hybridization......Page 245
Anisotropies of pteridine-containing sequences to examine protein binding......Page 247
Lifetimes, steady-state and time-resolved anisotropies of 3MI- and 6MI-containing sequences......Page 248
Probing the hairpin structure of an aptamer using 6MI......Page 250
Two-photon excitation of 6MAP......Page 252
Single molecule detection of 3MI......Page 255
Summary......Page 256
References......Page 257
Introduction......Page 261
Labeling Methods......Page 264
Diffusion Single-Pair FRET for RNA Based Systems......Page 266
Total Internal Reflection Fluorescence (TIRF) for RNA Based Systems......Page 268
Application 1: Folding of the Hairpin Ribozyme......Page 271
Application 2: Assembly of the Rev-RRE Complex......Page 275
References......Page 277
Using Fluorophore-Labeled Oligonucleotides to Measure Affinities of Protein-DNA Interactions......Page 281
Definitions of Fluorescence Anisotropy and Intensity......Page 282
Advantages of Fluorescence Measurements......Page 284
Disadvantages of Fluorescence Measurements......Page 285
Designing the Oligonucleotide......Page 286
Designing the Experiment......Page 294
Competition Assays for Determining Specificity......Page 297
References......Page 298
Identifying Small Pulsatile Signals within Noisy Data: A Fluorescence Application......Page 301
Methods......Page 302
AutoDecon procedure......Page 303
AutoDecon fitting module......Page 304
AutoDecon triage module......Page 305
AutoDecon combined modules......Page 306
Concordant secretion events......Page 307
Results......Page 308
Discussion......Page 312
References......Page 314
Determination of Zinc Using Carbonic Anhydrase-Based Fluorescence Biosensors......Page 315
Principles of CA-Based Zinc Sensing......Page 316
Transducing Zinc Binding as a Fluorescence Change......Page 318
"Free" Versus Bound Zinc Ion: Speciation......Page 323
Metal Ion Buffers......Page 325
Kinetics......Page 329
Applications: Ratiometric Determination of Free Zinc in Solution......Page 330
Preparation of Apocarbonic Anhydrase......Page 331
Intracellular Sensing with TAT Tag......Page 332
Intracellular Sensing with an Expressible CA Sensor......Page 333
References......Page 335
Instrumentation for Fluorescence-Based Fiber Optic Biosensors......Page 338
Introduction and Rationale for Fluorescence-Based Fiber Optic Sensors......Page 339
Basic Principles of Fiber Optics for Fluorescence Sensors......Page 340
Alignment of sensors......Page 343
Mounting and Alignment of the Instrument......Page 346
Standards for Ratiometric and Lifetime-Based Fiber Optic Fluorescence Sensors......Page 350
Construction of Fiber Optic Probes......Page 353
Zn2+ Probe......Page 356
Cu2+ Probe......Page 357
Use of Fiber Optic Probes for Discrete Samples......Page 359
Operating Issues: Noise, Background, Thermal Drift, and Mode Hopping......Page 360
Field and Shipboard Use......Page 361
Acknowledgments......Page 362
References......Page 363
Author Index......Page 365
Subject Index......Page 379