Principles of Fluorescence Spectroscopy

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528 ENERGY TRANSFER TO MULTIPLE ACCEPTORS IN ONE,TWO, OR THREE DIMENSIONS Figure 15.30. Relative donor quantum yield versus C/C 0 in one (1D), two (2D), and three (3D) dimensions. brane (acceptor per phospholipid), and for DNA (acceptor per base pair). For the membranes assume 70 Å 2 per phospholipid, and for DNA assume 3.4 Å per base pair. Use R 0 = 50 Å. Are these acceptor concentrations practical for proteins, membranes or nucleic acids? P15.4. Calculation of R 0 from the Extent of Donor Quenching: Figure 15.31 shows calculated donor decays for membrane-bound donors and acceptors. 50 The simulations were performed using 70 Å 2 per lipid mol- Figure 15.31. Calculated time-resolved decays of donor fluorescence for membrane-bound donors and acceptors. The area per lipid (PL) molecule was taken as 70 Å 2 per lipid. Revised from [50]. ecule and the acceptor densities (A/PL) shown in Figure 15.31. Calculate the R 0 value for this donor–acceptor pair.
16 The biochemical applications of fluorescence often utilize intrinsic protein fluorescence. Among biopolymers, proteins are unique in displaying useful intrinsic fluorescence. Lipids, membranes, and saccharides are essentially nonfluorescent, and the intrinsic fluorescence of DNA is too weak to be useful. In proteins, the three aromatic amino acids— phenylalanine, tyrosine, and tryptophan—are all fluorescent. These three amino acids are relatively rare in proteins. Tryptophan, which is the dominant intrinsic fluorophore, is generally present at about 1 mole% in proteins. The small number of tryptophan residues is probably the result of the metabolic expense of its synthesis. A protein may possess just one or a few tryptophan residues, facilitating interpretation of the spectral data. If all twenty amino acids were fluorescent then protein emission would be more complex. A valuable feature of intrinsic protein fluorescence is the high sensitivity of tryptophan to its local environment. Changes in the emission spectra of tryptophan often occur in response to conformational transitions, subunit association, substrate binding, or denaturation. These interactions can affect the local environment surrounding the indole ring. Tyrosine and tryptophan display high anisotropies that are often sensitive to protein conformation and the extent of motion during the excited-state lifetime. Also, tryptophan appears to be uniquely sensitive to collisional quenching, apparently due to a tendency of excited-state indole to donate electrons. Tryptophan can be quenched by externally added quenchers or by nearby groups within the proteins. There are numerous reports on the use of emission spectra, anisotropy, and quenching of tryptophan residues in proteins to study protein structure and function. A complicating factor in the interpretation of protein fluorescence is the presence of multiple fluorescent amino acids in most proteins. The environment of each residue is distinct and the spectral properties of each residue are generally different. However, the absorption and emission spectra of tryptophan residues in proteins overlap at most Protein Fluorescence usable wavelengths, and it is difficult to separate the spectral contributions of each tryptophan in a multi-tryptophan protein. Complex time-resolved intensity decays are found even for proteins that contain a single-tryptophan residue. Most single-tryptophan proteins display multi-exponential intensity decays. For this reason one cannot simply interpret the multiple decay times of a multi-tryptophan protein in terms of the individual tryptophan residues in a protein. As a further complicating factor, tryptophan displays complex spectral properties due to the presence of two nearly isoenergetic excited states, 1 L a and 1 L b . The electronic transitions display distinct absorption, emission, and anisotropy spectra, and are differently sensitive to solvent polarity. The complexity of indole photophysics has stimulated detailed studies of protein fluorescence, but has also inhibited interpretation of the data. Protein fluorescence can be complex, but considerable progress has been made in the past decade. The origin of the multi-exponential decay of tryptophan in water is now largely understood as due to the presence of rotational conformational isomers (rotamers). These rotamers have different orientations of the amino and carboxyl groups relative to the indole ring. Studies of single-tryptophan proteins have provided information on the spectral properties of tryptophan in unique environments. Widely different spectral properties have been observed for single-tryptophan proteins. The highly variable tryptophan quantum yields of proteins appear to be the result of nearby quenchers in the protein, which include lysine and histidine residues. Under appropriate conditions it appears that even amide groups in the peptide backbone can act as quenchers. The use of sitedirected mutagenesis has allowed creation of mutants that contain one instead of several tryptophan residues, insertion of tryptophan at desired locations in the protein, and modification of the environment around a tryptophan residue. Examination of single-tryptophan proteins and engineered proteins has provided a more detailed understanding of how 529
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Principles of Fluorescence Spectros
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Joseph R. Lakowicz Center for Fluor
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Preface The first edition of Princi
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x GLOSSARY OF ACRONYMS DNS dansyl o
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xii GLOSSARY OF ACRONYMS TRES time-
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xiv GLOSSARY OF MATHEMATICAL TERMS
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xvi CONTENTS 2.9.4. Conversion betw
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xviii CONTENTS 6. Solvent and Envir
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xx CONTENTS 10.4.4. Alignment of Po
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xxii CONTENTS 14.7.1. Simulations o
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xxiv CONTENTS 19.12. New Approaches
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xxvi CONTENTS 25.3. Optical Propert
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2 INTRODUCTION TO FLUORESCENCE Figu
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6 INTRODUCTION TO FLUORESCENCE inst
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10 INTRODUCTION TO FLUORESCENCE Fig
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12 INTRODUCTION TO FLUORESCENCE ns,
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14 INTRODUCTION TO FLUORESCENCE 1.7
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24 INTRODUCTION TO FLUORESCENCE due
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28 INSTRUMENTATION FOR FLUORESCENCE
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3 Fluorescence probes represent the
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PRINCIPLES OF FLUORESCENCE SPECTROS
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98 TIME-DOMAIN LIFETIME MEASUREMENT
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100 TIME-DOMAIN LIFETIME MEASUREMEN
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5 In the preceding chapter we descr
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6 Solvent polarity and the local en
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238 DYNAMICS OF SOLVENT AND SPECTRA
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278 QUENCHING OF FLUORESCENCE 8.1.
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280 QUENCHING OF FLUORESCENCE Figur
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282 QUENCHING OF FLUORESCENCE ly ev
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290 QUENCHING OF FLUORESCENCE relat
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298 QUENCHING OF FLUORESCENCE σ ar
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320 QUENCHING OF FLUORESCENCE 47. R
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322 QUENCHING OF FLUORESCENCE 113.
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328 QUENCHING OF FLUORESCENCE P8.3.
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330 QUENCHING OF FLUORESCENCE P8.11
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332 MECHANISMS AND DYNAMICS OF FLUO
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10 Measurements of fluorescence ani
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11 In the preceding chapter we desc
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12 In the preceding two chapters we
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444 ENERGY TRANSFER Figure 13.1. Fl
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446 ENERGY TRANSFER wavelength is e
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448 ENERGY TRANSFER Table 13.1. Cal
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450 ENERGY TRANSFER Figure 13.6. Ab
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452 ENERGY TRANSFER safe for many p
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454 ENERGY TRANSFER Figure 13.12. P
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456 ENERGY TRANSFER Figure 13.16. E
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458 ENERGY TRANSFER Figure 13.21. R
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460 ENERGY TRANSFER Figure 13.24. R
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462 ENERGY TRANSFER tiple acceptors
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464 ENERGY TRANSFER Figure 13.29. A
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466 ENERGY TRANSFER Figure 13.33. F
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468 ENERGY TRANSFER Table 13.3. Rep
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470 ENERGY TRANSFER 55. Clegg RM. 1
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472 ENERGY TRANSFER Tong AK, Jockus
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474 ENERGY TRANSFER Figure 13.39. O
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580 TIME-RESOLVED PROTEIN FLUORESCE
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608 MULTIPHOTON EXCITATION AND MICR
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19 Fluorescence sensing of chemical
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676 NOVEL FLUOROPHORES Figure 20.1.
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678 NOVEL FLUOROPHORES Figure 20.7.
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680 NOVEL FLUOROPHORES Figure 20.12
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698 NOVEL FLUOROPHORES 33. Acherman
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700 NOVEL FLUOROPHORES 103. Demas J
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702 NOVEL FLUOROPHORES 176. Montalt
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21 During the past 20 years there h
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22 Fluorescence microscopy is one o
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758 SINGLE-MOLECULE DETECTION Figur
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766 SINGLE-MOLECULE DETECTION small
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770 SINGLE-MOLECULE DETECTION Table
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786 SINGLE-MOLECULE DETECTION face
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788 SINGLE-MOLECULE DETECTION TCSPC
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790 SINGLE-MOLECULE DETECTION 53. H
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792 SINGLE-MOLECULE DETECTION Ke PC
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794 SINGLE-MOLECULE DETECTION Polar
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24 In the previous chapter we descr
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862 RADIATIVE-DECAY ENGINEERING: SU
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Appendix I Corrected Emission Spect
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Appendix II Fluorescence Lifetime S
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890 APPENDIX III P ADDITIONAL READI
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892 APPENDIX III P ADDITIONAL READI
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894 ANSWERS TO PROBLEMS A1.4. A. Th
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896 ANSWERS TO PROBLEMS Energy tran
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898 ANSWERS TO PROBLEMS Figure 4.65
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900 ANSWERS TO PROBLEMS expected to
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904 ANSWERS TO PROBLEMS where f is
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906 ANSWERS TO PROBLEMS [BSA] Obser
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908 ANSWERS TO PROBLEMS emission. T
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910 ANSWERS TO PROBLEMS dx rA A (
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912 ANSWERS TO PROBLEMS B. A covale
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914 ANSWERS TO PROBLEMS The α i va
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920 ANSWERS TO PROBLEMS CHAPTER 24
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Index A Absorption spectroscopy, 12
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