Principles of Fluorescence Spectroscopy

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488 TIME-RESOLVED ENERGY TRANSFER AND CONFORMATIONAL DISTRIBUTIONS OF BIOPOLYMERS be understood by considering the effects of incomplete acceptor labeling. This will result in the donor decay containing components due to both the D–A pairs, and from the donors that lack acceptors. The donor-alone molecules contribute to the measured emission greater than their mole fraction. For instance, suppose the donors are quenched tenfold by the linked acceptor, and that 10% of the donors lack acceptors. The donor-alone molecules will contribute tenfold more than their 10% mole fraction, so that the donoralone signal will be equal to that of the D–A pairs. Hence, a small fraction of underlabeling by acceptors can result in Figure 14.14. Flowchart for distance-distribution analysis. severe distortion of the donor decays. We have found that the presence of a small fraction of donor-alone molecules usually results in a failure of the Gaussian distance-distribution analysis to converge. This is a fortunate occurrence, and preferable to a convergent analysis that may be accepted as correct. We have less experience with the properties of the other distributions in the presence of a significant donor-alone population. The presence of underlabeling by the acceptor can be accounted for in the least-squares analysis. The donor intensity decay is given by
PRINCIPLES OF FLUORESCENCE SPECTROSCOPY 489 I DA(t) (1 f A)I 0 D ∑ i f AI 0 D ∞ 0 P(r) ∑ i α 0i exp(t/τ Di) αDi exp [ t τDi t ( τDi R0 r ) 6 ] (14.24) where f A is the fraction of the molecules labeled with acceptors. 29–30 While it is possible to correct for acceptor underlabeling, its presence results in decreased information in the donor decay kinetics, and quickly results in loss of resolution about the distance distribution. There seems to be strong correlation between f A , r, and hw during the leastsquares analysis. If the value of f A is not known, we regard 5% underlabeling by acceptors as the maximum consistent with reasonable confidence of the distance distribution. If f A is known, and not a variable in the analysis, then a higher level of underlabeling can be tolerated. Surprisingly, the extent of donor underlabeling is not important in the distance-distribution analysis using the donor emission. Molecules lacking the donor (that is, labeled only with acceptor) do not contribute to the donor decay. The presence of acceptor-only molecules is not important since the solutions are typically too dilute for transfer to acceptors not covalently linked to donors. 14.4.5. Effect of the Orientation Factor κ 2 As described in the previous chapter, the rate of energy transfer depends on the relative orientation of the donor emission dipole and the acceptor absorption dipole. Fortunately, for studies of distance distributions, the effect of κ 2 is likely to be small and use of the dynamic average κ 2 = 2/3 is reasonable. 31–33 This is because angular displacements of the donor and acceptor result in averaging the value of κ 2 towards 2/3. For a native protein one can expect the donors and acceptors to be relatively immobile during the excitedstate lifetime. This is less likely in the random-coil state, where the donor and acceptor, and the peptide itself, can be expected to rotate and diffuse. For molecules the size of fluorophores, the rotational correlation times are typically near 100 ps in water or when located on the surface of a macromolecule. The only mechanism by which κ 2 could affect the distance distribution is if the average D–A orientation were in some way correlated with the D–A distance. 31–32 Given the multitude of conformations accessible to random-coil peptides, this seems unlikely. In general, rotational probe motions occur more rapidly than translational diffusion, so that one can expect rotational averaging even when D–A translational diffusion is minimal. In general, it seems that distance distributions should be largely unaffected by κ 2 , especially where the solution is fluid enough to allow segmental motions of the probes relative to the biomolecule. 14.4.6. Acceptor Decays Information about the distance distribution is also contained in the acceptor decays, so that one may ask why we have stressed measurements of the donor decays. Although in principle either the donors or acceptor decays could be used to recover the distance distribution, 34–35 in most circumstances there is less information in the acceptor decay than in the donor decay. This can be seen by examining the absorption spectra of tryptophan (indole) and dansyl (Figure 13.9). The acceptors typically absorb light at wavelengths used to excite the donors. As a result, the acceptor is excited by two routes: by direct excitation and by RET from the donor. Only the latter portion of the acceptor emission contains information on the distance distribution. Typically, this is the minor fraction of the acceptor intensity, so that most of the acceptor emission is due to the intrinsic decay of the directly excited acceptor. Because of this fact any analysis of the acceptor-decay kinetics must consider directly excited acceptors. Depending on the donor and acceptor emission spectra it may also be necessary to consider contributions of the donor emission at the wavelength chosen to observe the acceptor. In principle an analysis correcting for donor emission and a directly excited acceptor can be performed, but such analyses rarely appear in the literature. If one can measure the acceptor-decay kinetics due only to RET it will display the unique properties of an excited-state reaction. In the time domain the characteristics of an excited-state reaction are a rise time in the timedependent intensities, and a negative pre-exponential factor in the multi-exponential analysis. In the frequency domain the phase angles of the acceptor can exceed 90E. These characteristic features are the result of the acceptor being excited by the donor, rather than directly. These features can be hidden by spectral overlap of the donor and acceptor emission spectra and by a directly excited acceptor.
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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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Page 453 and 454: PRINCIPLES OF FLUORESCENCE SPECTROS
Page 461 and 462: 444 ENERGY TRANSFER Figure 13.1. Fl
Page 463 and 464: 446 ENERGY TRANSFER wavelength is e
Page 465 and 466: 448 ENERGY TRANSFER Table 13.1. Cal
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Page 479 and 480: 462 ENERGY TRANSFER tiple acceptors
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Page 485 and 486: 468 ENERGY TRANSFER Table 13.3. Rep
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Page 503: PRINCIPLES OF FLUORESCENCE SPECTROS
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578 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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