CONTENTS xVii 7 6c.Distinction between Solvent 8.11.A.Quenching Due to Specific Binding Relaxation and Formation of Interactions 255 Rotational isomers 229 8.11.B.Binding of Substrates to Ribozymes 256 7.7.Comp arison of TRES and DAS 230 ation react ions and Quenching )57 78 Red-fdge frcitation shifts 231 812 ching 757 7.9 erspectives of Solvent Dynamics. 233 8.13.Q of Ph osphores 058 233 50 Problems 236 Probl 264 8.Quenching of Fluorescence 9.Advanced Topics in Fluorescence Quenching 8.1.Quenchers of Fluorescence 238 9.1.Quenching in Membr 267 8.2.Theory of Collisional Quenching. 239 9.1.A A of Memh rane Probes to 8.2.A.Derivation of the Stern-Volmer and Lipid-So Quenchers 267 Eouation 240 8.2.B.Interpretation of the Bimolecular 9.1.B.Quenching of Membrane Probes Using Localized Quenchers 270 Quenching Const 241 8.3.Theor 242 9.1.C.Parallax Quenching in Membranes.272 84 .273 nic and s Static Que 9.1.D.Boundary Lipid Quenching. 9.1.E.Effect of Lipid-Water Partitioning on and Dynan e ching Ouenching 74 ons fr om the olmer Equation 9.2.Diffusion in Membranes 276 Que nchi ing Sphere o Action 244 92 A Ouasi-Thre Dimensional diffusion 8.6.A.Derivation of the Ouenching Sphere of Memh Action 245 9.2.B al Diffus on in Membranes 8.7.Effects of Steric Shielding and Charge on Quenching 9.3.Quenc hing Effic ey 245 9.3.A.Steric Shielding Effects in Quenching 279 8.7.A.Accessibility of DNA-Bound Probes 9.4.Transient Effects in Quenching 280 to Quenchers 246 9.4.A.Experimental Studies of Transient 8.7.B. ching of Eth adenine Effects. .281 at 9.4.B.Distance-Dependent Quenching in 8.8.Fracti sibility toQuencher 24 285 8.8.A.Modified Stern-Volmer Plots 248 References 286 8.8.B.Experimental Considerations in Problems. 289 Quenching 249 8.9.Applications of Quenching to Proteins 249 8.9A.Fractional Accessibility of Tryptophan 10.Fluorescence Anisotropy Residues in Endonuclease m 8.9.B Effect of Confor onal Changes on 10.1.Definition of Fluorescence Anisotropy.291 Tryptophan Ac ibility 250 10.1.A.Origin of the Definitions of 8.9.C.Quenching of the Multiple Decay Polarization and Anisotropy 292 Times of Proteins. 250 10.2.Theory for Anisotropy 293 89.D Effects of ouenchers on Proteins 251 10.2.A.Excitation Photoselection of 8.9E Protein Folding of Colicin El 51 294 8.10.0 nching-Resolved Emission Spectra 252 10.3.Excitatic 295 810.A. ore mixture 252 0.3.A of Ele tronic States from 810.B Quenc Polariz Spectra 297 t the let Repres 253 10.4. 29 8.11.Quenching and Association Reactions. 255 10.4.A.L-Format or Single-Channel Method 298
PRINCPLES OF FLUORESCENCE SPECTROSCOPY 10.4.B.T-Format or Two-Channel Anisotropies 299 11.2.A. Time-Domain Anisotropy Data.325 10.4.C.Comparison ofT-Format and L-Format 11.2.B.Value of ro. .327 Measurements 300 11.3.Analysis of Frequency-Domain Anisotropy 104 D Alignment of Polarizers 300 Decays .327 10.4.E.Magic-Angle Polarizer Conditions 301 11.4.Anisotropy Decay Laws 328 10.4.F.Why Is the Total Intensity Equal to 11.4.A.Nonspherical Fluorophores 328 421.7 I4 B Hindered Rotors 329 10.4.G.Efc ct of Radiationless Energy 11.4.C.Segmental Mobility of a Biopolymer- Ira on the 302 nd可u 320 10.4.H.TrivialCa Anisotropy es of Depolarization 10.4.I.Factors Affe ting the Anisotropy·. 303 10.5.Effects of Rotational Diffusion on Fluorescence 1.5 ed Rot 33 Anisotropies:The Perrin Equation . 303 11.6 ain Anisotropy De 333 10.5.A.The Perrin Equation-Rotational 11.6.A.Intrinsic Tryptophan Anisotropy Decay Motions of Proteins 304 of Liver Alcohol Dehydrogenase. 333 10.5.B.Examples of Perrin Plots 306 11.6.B.Phospholipase A2. 334 10.6.Perrin Plots of Proteins 307 11.6.C.Domain Motions of Immunoglobulins 334 10.6.A.Binding of tRNA to tRNA Synth tase 307 11.6.D.Effects of Free Probe on Anisotropy 10.6.B.Molecular Chape 335 (GroEL) 307 117.Frequency-Domain Anisotropy Decays of 10.6.c rrin of f an F Immuno 33g globulin Fragment 117A yoglobi A Rigid Roto 335 10.7.Protei Association Reactions 308 11.7.B. on and 10.7.A.Peptide Binding to Calmodulin 308 ays 336 10.7.B.Binding of the Trp Repressor to DNA 309 11.7.C.Picosecond Rotational Diffusion of 10.7.C.Melittin Association Detected from 30g Oxytocin···· ，。 336 Homotransfer 11.8.Microsecond Anisotropy Decays. 337 10.8.Anisotropy of Membrane-Bound Probes 310 11.8.A.Phosphorescence Anisotropy Decays.337 10.9.Lifetime-Resolved Anisotropies. 310 11.8.B.Long-Lifetime Metal-Ligand 10.9.A.Effect of Segm ental motion on the 337 in Plot 31 11.9.Anisotropy Decays of Nucleic Acids 338 10.10 Multiplication of 11.9.A.Hydrody s of DNA Oligomers 339 312 340 10.11 11.10. racte rization of a New mbrane Probe 341 g 312 Re 342 10.12 on Moments 313 Pro 345 10.12.A.Anisotropy of Planar Fluorophores with High Symmetry·· 314 10.13.Anisotropies with Multiphoton Excitation. 315 12. Advanced Anisotropy Concepts 10.13.A.Excitation Photoselection for Two-Photon Excitation. 315 12.1.Rotational Diffusion of Nonspherical 10.13.B.Two-Photon Anisotropy of DPH 31 Molecules-An Overvicw. 347 Refer 316 12.1.A.Anisotropy Decays of Ellipsoids. 348 Problems 318 12.2.Ellipsoids of Revolution 349 12.2.A.Simplified Ellipsoids of Revolution 349 12.2.B.Intuitive Description of an Oblate 11.Time-Dependent Anisotropy Decays 351 12.2.C.Rotational Co elation Times for nalysofm Domain Anisoopy Deca 3 35 Anisotropy Decay Analysis 325 12.2.D. Stick versus Slip 353
CONTENTS xix 12.3.Complete Theory for Rotational Diffusion of 13.4.Energy Transfer in Membranes 382 Ellipsoids 354 13.4.A.Lipid Distributions around Gramicidin 384 12.4.Time-Domain Studies of Anisotropic Rotational 13.4 B.Distance of Closest Approach in Diffusion 354 385 12.5.Frequency-Domain Studies of Anisotropic 134CM nand Lipid Exchange 206 Rotational diffusion 35513.5.Bn ansfer in Solu 206 12.6.Global Anisotropy Decay Analysis with on-F nced Energy Transfer 387 multiwavelength excitation 357 13.6 300 P tive Ro Values 12.7.Global Anisotropy Decay Analysis with es Collisional Quenching 359 Problems 391 12.7.A.Application of Quenching to Protein Anisotropy Decays 360 128 DNA 61 12.9. ciated Aniso 362 14.Time-Resolved Energy Transfer and 12.9.A.Theory for As Conformational Distributions of Biopolymers cays 363 12.9.B.Time-Domain Measurements of Associated Anisotropy Decays. 364 14.1.Distance Distributions ,3 12.9.C.Frequency-Domain Measurements of 14.2.Distance Distributions in Peptides.398 Associated Anisotropy Decays 364 14.2.A.Comparison for a Rigid and a References 365 Flexible Hexapeptide 398 Problems 366 14.2.B.Cross-fitting Data to Exelude Alternative Models 300 14D。n r Decay without RET 400 142.D.E t of Concentration of the D-A 13.Energy Transfer 14.3.Distanc Distribut 13.1.Theory of Energy Transfer fora ons in Proteins 48 Distance istributions in Melittin 401 368 14.3.B.Distance Distribution Analysis with 13.1.A.Orientation Factor K 371 Frequency-Domain Data. 404 13.1.B.Dependence of the Transfer Rate on 14.3.C.Distance Distributions from Distance (r),the Overlap Integral () Time-Domain Measurements 406 and K2 373 14.3.D.Analysis of Distance Distribution 13.1.C.Homotransfer and Heterotransfer 373 in Me d00 13.2.Distance Measu ents Using ReT 374 14.3.E.Domain Motion in Protein 40g 13.2.A.Distance Measure ents in a-Helical 4.3.F Dis Distribu Melittin 374 144 13.2.B the Possible Range of i45 of Diffusio air 411 Dis 375 14.5.A Simulations of RET for a Flexible 13.2.C.Protein Folding Measured by RET 376 D-A Pair 411 13.2.D.Orientation of a Protein-Bound Peptide 377 14.5.B.Experimental Measurement of D-A 13.3.Use of RET to Measure Macromolecular Diffusion for a Linked D-A Pair .412 Associations. 378 14.5.C.x Surfaces and Parameter Resolution 413 13.3.A.Dissociation of the Catalytic and 14.5.D.Diffusion and Apparent Distance Regulatory subunits of a protein 415 Kinase 378 14.5.E.RET and Diff Motions in 13 3B.RETCalcium Indicators 379 polymer 416 13.3.C Kinetics of DNA 14.6.Distan in Nucleic Acids 416 380 1 Hel DNA 13.3.D Energy Tra ncy from 6.B. r-way Holliday Junction in DNA Enhanced Acceptor Fluorescence 381 14.7.Other Considerations 418
PRINCIPLES OF FLUORESCENCE SPECTROSCOPY 14.7.A.Acceptor Decays. 418 16.1.D.Ground-State Complex Formation by 14.7.B.Effects of Incomplete Labeling. 418 Tyrosine 。 451 14.7.C.Effect of the Orientation Factor K2 419 16.1.E.Excimer Formation by Phenylalanine 452 14.8.Distance Distributions from Steady-State Data 419 16.2.General Features of Protein Fluorescence .452 14.8.A.D-A Pairs with Different Ro Values 41 16.3.Tryptophan Emission in an Apolar Protein 14.8.B Changing ra by ouenching 420 Environment 454 14.9.Applications of Time-Resolved RET 420 16.3.A.Site-Directed Mutagenesis of a 14.9.A.DNA Hybridization 40 Single. 454 14.10.Conclusio 421 16.3.B ctra of Azurins with 47 Problems e or Tw Tryptophan Residues 16.4.Energy Trans er in Protein 16.4.A.Tyrosine-to-Tryptophan Energy Transfer in Interferon-y. 456 15.Energy Transfer to Multiple 16.4.B.Ouantitation of RET Efficiencies in Acceptors,in One,Two,or Three Proteins 457 Dimensions 16.4.C.Energy Transfer Detected by Decr ases in Anisotropy 459 15.1.RET in Three Dim 16.4.D.Phenylalanine-to-Tyr osine Energy ons 426 15.1.A.Effect of Diffusion on RET with Unlinked Donors and Acceptors·· 2 1.5.Quenching of Tryptophan 15.2.Effect of Dimensionality on RET. 461 429 165.A 15.2.A.Experimental RET in Two Effect of Emission Maximum on Quenching 461 Dimensions 430 15.2.B.Experimental RETin One Dimension 16.5.B.Fractional Accessibility to 43 15.3.Energy Transfer in Restricted Geom 434 Quenching in Multitryptophan Proteins 463 15.3.A.Effect of an Excluded Are on 16.5.C.Resolution of Emission Spectra by imensions 139 Quenching 464 15.4A 16.6.Association Rea ons of Proteins 465 on of an Acceptor in Lipid Vesicles 437 16.6.A Self-Association of Melittin and 15.4.B.Location of Retinal in Rhodopsin 465 438 16.6.B Ligand Binding to Proteins Disk Membranes. 15.4.C.Forster Transfer versus Exchange 16.6.C.Correlation of Emission Maxima, Anisotropy.and Quenching Constant Interactions 439 466 15.5.Conclusions. 440 for Tryptophan Residues References 16.6.D.Calmodulin:Resolution of the Four 440 Problems Calcium Binding Sites Using T an-Cor eM. 468 16.7. ral Properties of Geneti ically Engineered ein 469 16.Protein Fluorescence 16.7.A Protein Tyrosyl Phosphatase Simple Two-Tryptophan Protein 469 16.1.Spectral Properties of the Aromatic Amino 16.7.B.Human Tissue Factor Contains Acids. 46 Nonfluorescent Tryptophan Residues 470 16.1.A.Excitation Polarization Spectra of 16.7.C.Barnase-A Three-Tryptophan Tyrosine and Tryptophan 447 Protein 470 16.1.B.Solvent Effects on Tryptophan 16.7D.Substrate Binding and Site-Directed Emission Spectra 449 esis 472 16.1.C.Excited-State Ionization of 16.7.E.Site-Din ed Mu age nesis of ine 450 Tyrosine Prote 472
CONTENTS xxi 16.8.Protein Folding 473 17.8.B.Immunophilin FKBP59-Quenching 16 8 a protein engineering of mutant of Tryptophan Fluorescence by ribonucleas e for Folding Phenylalanine 503 474 17.8.C _Resolution of the l6.8B Trp Re D: of Tryptophan Residues in Each e-Din 504 Domain 474 17.8.D.Aspartate Transcarbamylas 16.8.C.Emission Spectra of Native and Noninteracting Tryptophan Residues 504 Denatured Proteins 475 17.8.E.Thermophilic B-Glycosidase- 16.8.D.Folding of Lactate Dehydrogenase 475 Multi-tryptophan Protein 505 16.9. Tryptophan Analogs 476 17.8.F.Heme Proteins Display Useful 16.10.Multiphoton Excitation of Proteins. 479 Intrinsic Fluorescence 506 16.11.The Challenge of Protein Fluorescence 480 17.0 Phosphorescence of Proteins S08 References 481 17.10.Pe P光 ctives on protein Fluorescence 500 Problems 485 Re 510 Proble 514 17.Time-Resolved Protein Fluorescence 18.Excited-State Reactions 17.1.Intensity Decays of Tryptophan-The er Model 488 18 1 Examnles of Excited-State reactions 515 17.2.Time-Resolved Intensity Decays of 18.1.A Excited-State Iot ion of Naphthol 516 and T 489 18.2. 518 Decay-A iated Emission Spectra 18.2.A.Steady-St e Fl escence of a of Tryptophan. 490 Two-State Reaction . 518 17.2.B.Intensity Decays of Neutral 18.2.B.Time-Resolved Decays for the Tryptophan Derivatives. Two-State Model .518 17.2.C.Intensity Decays of Tyrosine and Its 18 3.Time-Domain Studies of Naphthol Neutral derivatives 491 Dissociation .519 17.3.Intensity Decays of Proteins 492 18.3.A.Differential-Wavelength Methods.520 17.4.Effects of Protein Structure on the Intensity 18.4.Analysis of Excited-State Reactions by and Anisotropy Decay of Ribonuclease T 493 Phase-Modulation Fluorometry 521 8.4.A Effect of an excited-State reaction e to Wat 493 nt Phase and 17.4.B Can 523 Result in Complex Intensity and 18.4.B Wave ngth ependent Phase and Anisotropy Decays. Modulation Values for an 17.5.Anisotropy Decays of Proteins 496 Excited-State Reaction. 524 17.5.A.Anisotropy Decays of Melittin 497 18.5.Frequency-Domain Measurement of Excimer 17.5.B.Protein Anisotropy Decays as Formation .526 Observed in the frequency Domain 498 18.6.Biochemical Examples of Excited-State 17.6.Time-Dependent Spectral Relaxation in Reactions .527 Proteins 498 18 6A.Excited-State Tautomerization of 17.7.De -Associated Emission Spectra of 7-Azaindole 527 499 18.6.B.By e of a memb rane-Bound 17.8.Time-Resolved Studies of Representative lesterolAnalog,·· 2 501 17.8A anexin V-A Calci 18.7.Conclusion n-Sensitive 529 Single-Tryptophan Protein 501 530