Fluorescence resonance energy transfer (FRET) is transfer of the excited-state energy from the initially excited donor (D) to an acceptor (A). The donor molecules typically emit at shorter wavelengths which overlap with the absorption spectrum of the acceptor. Energy transfer occurs without the appearance of a photon and is the result of long-range dipole-dipole interactions between the donor and acceptor. The term resonance energy transfer (RET) is preferred because the process does not involve the appearance of a photon. The rate of energy transfer depends upon the extent of spectral overlap of the emission spectrum of the donor with the absorption spectrum of the acceptor, the quantum yield of the donor, the relative orientation of the donor and acceptor transition dipoles, and the distance between the donor and acceptor molecules. The distance dependence of RET has resulted in its widespread use to measure distances between donors and acceptors.


Energy Transfer Fluorescence Resonance Energy Transfer Transfer Efficiency Phosphatidic Acid Tryptophan Residue 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Förster, Th., 1948, Intermolecular energy migration and fluorescence, Ann. Phys. 2:55–75. Translated by R. S. Knox, Department of Physics and Astronomy, University of Rochester, Rochester, NY 14627.Google Scholar
  2. 2.
    Stryer, L., 1978, Fluorescence energy transfer as a spectroscopic ruler, Annu. Rev. Biochem. 47: 819–846.CrossRefGoogle Scholar
  3. 3.
    Steinberg, I. Z., 1971, Long-range nonradiative transfer of electronic excitation energy in proteins and polypeptides, Annu. Rev. Biochem. 40: 83–114.CrossRefGoogle Scholar
  4. 4.
    Clegg, R. M., 1996, Fluorescence resonance energy transfer, in Fluorescence Imaging Spectroscopy and Microscopy, X. F. Wang and B. Herman (eds.), John Wiley Sons, New York, pp. 179–252.Google Scholar
  5. 5.
    Fung, B. K. K., and Stryer, L., 1978, Surface density determination in membranes by fluorescence energy transfer, Biochemistry 17: 5241–5248.CrossRefGoogle Scholar
  6. 6.
    Weller, A., 1974, Theodor Forster, Ber. Bunsen-Ges. Phys. Chem. 78: 969–971.Google Scholar
  7. 7.
    Gordon, M., and Ware, W. R. (eds.), 1975, The Exciplex, Academic Press, New York.Google Scholar
  8. 8.
    Dale, R. E., Eisinger, J., and Blumberg, W. E., 1979, The orienta-tional freedom of molecular probes. The orientation factor in intramolecular energy transfer, Biophys. J. 26:161–194. Erratum 30: 365 (1980).Google Scholar
  9. 9.
    Dale, R. E., and Eisinger, J., 1975, Polarized excitation energy transfer, in Biochemical Fluorescence, Concepts, Vol. 1. R. F. Chen and H. Edelhoch (eds.), Marcel Dekker, New York, pp. 115–284.Google Scholar
  10. 10.
    Dale, R. E., and Eisinger, J., 1974, Intramolecular distances determined by energy transfer. Dependence on orientational freedom of donor and acceptor, Biopolymers 13: 1573–1605.CrossRefGoogle Scholar
  11. 11.
    Cheung, H. C., 1991, Resonance energy transfer, in Topics in Fluorescence Spectroscopy, Vol. 2, Principles, J. R. Lakowicz (ed.), Plenum Press, New York, pp. 127–176.Google Scholar
  12. 12.
    Wu, P., and Brand, L., 1994, Review—resonance energy transfer: Methods and applications, Anal. Biochem. 218: 1–13.CrossRefGoogle Scholar
  13. 13.
    Dos Remedios, C. G., and Moens, P. D. J., 1995, Fluorescence resonance energy transfer spectroscopy is a reliable “ruler” for measuring structural changes in proteins, J. Struct. Biol. 115: 175–185.CrossRefGoogle Scholar
  14. 14.
    Haas, E., Katchalski-Katzir, E., and Steinberg, I. Z., 1978, Effect of the orientation of donor and acceptor on the probability of energy transfer involving electronic transitions of mixed polarizations, Biochemistry 17: 5064–5070.CrossRefGoogle Scholar
  15. 15.
    Latt, S. A., Cheung, H. T., and Blout, E. R., 1965, Energy transfer. A system with relatively fixed donor-acceptor separation, J. Am. Chem. Soc. 877: 996–1003.Google Scholar
  16. 16.
    Stryer, L., and Haugland, R. P., 1967, Energy transfer: A spectroscopic ruler, Proc. Natl Acad. Sci. U.S.A. 58: 719–726.CrossRefGoogle Scholar
  17. 17.
    Gabor, G., 1968, Radiationless energy transfer through a polypeptide chain, Biopolymers 6: 809–816.CrossRefGoogle Scholar
  18. 18.
    Haugland, R. P., Yguerabide, J., and Stryer, L., 1969, Dependence of the kinetics of singlet-singlet energy transfer on spectral overlap, Proc. Natl Acad. Sci. U.S.A. 63: 23–30.CrossRefGoogle Scholar
  19. 19.
    Horrocks, W. DeW., Holmquist, B., and Vallee, B. L., 1975, Energy transfer between terbium(III) and cobalt(II) in thermolysin: A new class of metal-metal distance probes, Proc. Natl Acad. Sci. U.S.A. 72: 4764–4768.CrossRefGoogle Scholar
  20. 20.
    Johnson, I. D., Kang, H. C., and Haugland, R. P., 1991, Fluorescent membrane probes incorporating dipyrrometheneboron difluoride fluorophores, Anal Biochem. 198: 228–237.CrossRefGoogle Scholar
  21. 21.
    Hemmila, I. A. (ed.), 1991, Applications of Fluorescence in Immunoassays, John Wiley Sons, New York. (See p. 113.)Google Scholar
  22. 22.
    Kawski, A., 1983, Excitation energy transfer and its manifestation in isotropic media, Photochem. Photobiol. 38: 487–504.CrossRefGoogle Scholar
  23. 23.
    Selvin, P. R., 1995, Fluorescence resonance energy transfer, Methods Enzymol. 246: 300–334.CrossRefGoogle Scholar
  24. 24.
    Lakowicz, J. R., Gryczynski, I., Wiczk, W., Laczko, G., Prendergast, F. C., and Johnson, M. L., 1990, Conformational distributions of melittin in water/methanol mixtures from frequency-domain measurements of nonradiative energy transfer, Biophys. Chem. 36: 99–115.CrossRefGoogle Scholar
  25. 25.
    Faucon, J. F., Dufourca, J., and Lurson, C., 1979, The self-association of melittin and its binding to lipids, FEBSLett. 102: 187–190.CrossRefGoogle Scholar
  26. 26.
    Goto, Y., and Hagihara, Y., 1992, Mechanism of the conformational transition of melittin, Biochemistry 31: 732–738.CrossRefGoogle Scholar
  27. 27.
    Bazzo, R., Tappin, M. J., Pastore, A., Harvey, T. S., Carver, J. A., and Campbell, I. D., 1988, The structure of melittin: A *H-NMR study in methanol, Eur. J. Biochem. 173: 139–146.CrossRefGoogle Scholar
  28. 28.
    Lakowicz, J. R., Gryczynski, I., Cheung, H. C., Wang, C.-K., Johnson, M. L., and Joshi, N., 1988, Distance distributions in proteins recovered by using frequency-domain fluorometry. Applications to troponin I and its complex with troponin C, Biochemistry 27: 9149–9160.CrossRefGoogle Scholar
  29. 29.
    Cai, K., and Schircht, V., 1996, Structural studies on folding intermediates of serine hydroxymethyltransferase using fluorescence resonance energy transfer, J. Biol Chem. 271: 27311–27320.CrossRefGoogle Scholar
  30. 30.
    Chapman, E. R., Alexander, K., Vorherr, T., Carafoli, E., and Storm, D. R., 1992, Fluorescence energy transfer analysis of calmodulin-peptide complexes, Biochemistry 31: 12819–12825.CrossRefGoogle Scholar
  31. 31.
    Adams, S. R., Bacskai, B. J., Taylor, S. S., and Tsien, R. Y., 1993, Optical probes for cyclic AMP, in Fluorescent and Luminescent Probes for Biological Activity, W. T. Mason (ed.), Academic Press, New York, pp. 133–149.Google Scholar
  32. 32.
    Johnson, D. A., Leathers, V. L., Martinez, A.-M., Walsh, D. A., and Fletcher, W. H., 1993, Biomedical example: Use of FRET to measure subunit associations of the regulation (R) and catalytic (C) subunits of a protein kinase, Biochemistry 32: 6402–6410.CrossRefGoogle Scholar
  33. 33.
    Guptasarma, P., and Raman, B., 1995, Use of tandem cuvettes to determine whether radiative (trivial) energy transfer can contaminate steady-state measurements of fluorescence resonance energy transfer, Anal. Biochem. 230: 187–191.CrossRefGoogle Scholar
  34. 34.
    Romoser, V. A., Hinkle, P. M., and Persechini, A., 1997, Detection in living cells of Ca -dependent changes in the fluorescence emission of an indicator composed of two green fluorescent protein variants linked by a calmodulin-binding sequence, J. Biól. Chem. 272: 13270–13274.CrossRefGoogle Scholar
  35. 35.
    Miyawaki, A., Llopis, J., Heim, R., McCaffery, J. M., Adams, J. A., Ikura, M., and Tsien, R. Y., 1997, Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin, Nature 388: 882–887.CrossRefGoogle Scholar
  36. 36.
    Cardullo, R. A., Agrawal, S., Flores, C., Zamechnik, P. C., and Wolf, D. E., 1988, Detection of nucleic acid hybridization by nonradiative fluorescence resonance energy transfer, Proc. Natl Acad. Sci. U.S.A. 85: 8790–8794.CrossRefGoogle Scholar
  37. 37.
    Parkhurst, K. M., and Parkhurst, L. J., 1996, Detection of point mutations in DNA by fluorescence energy transfer, J. Biomed. Opt. 1: 435–441.CrossRefGoogle Scholar
  38. 38.
    Morrison, L. E., and Stols, L. M., 1993, Use of FRET to measure association of DNA oligomers, Biochemistry 32: 3095–3104.CrossRefGoogle Scholar
  39. 39.
    Ghosh, S. S., Eis, P. S., Blumeyer, K., Fearon, K., and Millar, D. P., 1994, Real time kinetics of restriction endonuclease cleavage monitored by fluorescence resonance energy transfer, Nucleic Acids Res. 22: 3155–3159.CrossRefGoogle Scholar
  40. 40.
    Le Bonniec, B. F., Myles, T., Johnson, T., Knight, C. G., Tapparelli, C., and Stones, S. R., 1996, Characterization of the F2 and specificities of thrombin using fluorescence-quenched substrates and mapping of the subsites by mutagenesis, Biochemistry 35: 7114–7122.CrossRefGoogle Scholar
  41. 41.
    Lee, S. P., Censullo, M. L., Kim, H. G., Knutson, J. R., and Han, M. K., 1995, Characterization of endonucleolytic activity of HIV-1 integrase using a fluorogenic substrate, Anal Biochem. 227: 295–301.CrossRefGoogle Scholar
  42. 42.
    Mayayoshi, E. D., Wang, G. T., Krafft, G. A., and Erickson, J., 1990, Novel fluorogenic substrates for assaying retroviral proteases by resonance energy transfer, Science 247: 954–958.CrossRefGoogle Scholar
  43. 43.
    Mitra, R. D., Silva, C. M., and Youvan, D. C., 1996, Fluorescence resonance energy transfer between blue-emitting and red-shifted excitation derivatives of the green fluorescent protein, Gene 173: 13–17.CrossRefGoogle Scholar
  44. 44.
    Perkins, T. A., Wolf, D. E., and Goodchild, J., 1996, Fluorescence resonance energy transfer analysis of ribozyme kinetics reveals the mode of action of a facilitator oligonucleotide, Biochemistry 35: 16370–16377.CrossRefGoogle Scholar
  45. 45.
    Lee, P., and Han, M. K., 1997, Fluorescence assays for DNA cleavage, Methods Enzymol 278: 343–361.CrossRefGoogle Scholar
  46. 46.
    Bilderback, T., Fulmer, T., Mantulin, W. W, and Glaser, M., 1996, Substrate binding causes movement in the ATP binding domain of Escherichia coli adenylate kinase, Biochemistry 35: 6100–6106.CrossRefGoogle Scholar
  47. 47.
    Epe, B., Steinhauser, K. G., and Woolley, P., 1983, Theory of measurement of Fòrster-type energy transfer in macromolecules, Proc. Natl Acad. Sci. U.S.A. 80: 2579–2583.CrossRefGoogle Scholar
  48. 48.
    Clegg, R. M., 1992, Fluorescence resonance energy transfer and nucleic acids, Methods Enzymol. 211: 353–388.CrossRefGoogle Scholar
  49. 49.
    Clegg, R. M., Murchie, A. I. H., and Lilley, D. M., 1994, The solution structure of the four-way DNA junction at low-salt conditions: A fluorescence resonance energy transfer analysis, Biophys. J. 66: 99–109.CrossRefGoogle Scholar
  50. 50.
    Berman, H. A., Yguerabide, J., and Taylor, P., 1980, Fluorescence energy transfer on acetylcholinesterase: Spatial relationships between peripheral site and active center, Biochemistry 19: 2226–2235.CrossRefGoogle Scholar
  51. 51.
    Pedersen, S., Jorgensen, K., Baekmark, T. R., and Mouritsen, O. G., 1996, Indirect evidence for lipid-domain formation in the transition region of phospholipid bilayers by two-probe fluorescence energy transfer, Biophys. J. 71: 554–560.CrossRefGoogle Scholar
  52. 52.
    Estep, T. N., and Thomson, T. E., 1979, Energy transfer in lipid bilayers, Biophys. J. 26: 195–207.CrossRefGoogle Scholar
  53. 53.
    Wolber, P. K., and Hudson, B. S., 1979, An analytical solution to the Forster energy transfer problem in two dimensions, Biophys. J. 28: 197–210.CrossRefGoogle Scholar
  54. 54.
    Dewey, T. G., and Hammes, G. G., 1980, Calculation of fluorescence resonance energy transfer on surfaces, Biophys. J. 32: 1023–1035.CrossRefGoogle Scholar
  55. 55.
    Shaklai, N., Yguerabide, J., and Ranney, H. M., 1977, Interaction of hemoglobin with red blood cell membranes as shown by a fluorescent chromophore, Biochemistry 16: 5585–5592.CrossRefGoogle Scholar
  56. 56.
    Snyder, B., and Freire, E., 1982, Fluorescence energy transfer in two dimensions, Biophys. J. 40: 137–148.CrossRefGoogle Scholar
  57. 57.
    Bastiaens, P., de Beus, A., Lacker, M., Somerharju, P., Vauhkonen, M., and Eisinger, J., 1990, Resonance energy transfer from a cylindrical distribution of donors to a plane of acceptors, Biophys. J. 58: 665–675.CrossRefGoogle Scholar
  58. 58.
    Yguerabide, J., 1994, Theory for establishing proximity relations in biological membranes by excitation energy transfer measurements, Biophys. J. 66: 683–693.CrossRefGoogle Scholar
  59. 59.
    Dewey, T. G., 1991, Fluorescence energy transfer in membrane biochemistry, in Biophysical and Biochemical Aspects of Fluorescence Spectroscopy, T. G. Dewey (ed.), Plenum Press, New York, pp. 197–230.Google Scholar
  60. 60.
    Dobretsov, G. E., Kurek, N. K., Machov, V. N., Syrejshehikova, T. I., and Yakimenko, M. N., 1989, Determination of fluorescent probes localization in membranes by nonradiative energy transfer, J. Bio-chem. Biophys. Methods 19: 259–274.CrossRefGoogle Scholar
  61. 61.
    Tweet, A. G., Bellamy, W. D., and Gaines, G. L., 1964, Fluorescence quenching and energy transfer in monomolecular films containing chlorophyll, J. Chem. Phys. 41: 2068–2077.CrossRefGoogle Scholar
  62. 62.
    Loura, L. M. S., Fedorov, A., and Prieto, M., 1996, Resonance energy transfer in a model system of membranes: Application to gel and liquid crystalline phases, Biophys. J. 71: 1823–1836.CrossRefGoogle Scholar
  63. 63.
    Stryer, L. (ed.), 1995, Biochemistry, 4th ed., W. H. Freeman and Company, New York. (See p. 274.)Google Scholar
  64. 64.
    Wang, S., Martin, E., Cimino, J., Omann, G., and Glaser, M., 1988, Distribution of phospholipids around gramicidin and D-p-hydroxy-butyrate dehydrogenase as measured by resonance energy transfer, Biochemistry 27: 2033–2039.CrossRefGoogle Scholar
  65. 65.
    Shahrokh, Z., Verkman, A. S., and Shohet, S. B., 1991, Distance between skeletal protein 4.1 and the erythrocyte membrane bilayer measured by resonance energy transfer, J. Biol. Chem. 266: 12082–12089.Google Scholar
  66. 66.
    McCallum, C. D., Su, B., Neuenschwander, P. F., Morrissey, J. H., and Johnson, A. E., 1997, Tissue factor positions and maintains the factor Vila active site far above the membrane surface even in the absence of the factor Vila Gla domain, J. Biol. Chem. 272: 30160–30166.CrossRefGoogle Scholar
  67. 67.
    Davenport, L., Dale, R. E., Bisby, R. H., and Cundall, R. B., 1985, Transverse location of the fluorescent probe 1,6-diphenyl-1,3,5- hexatriene in model lipid bilayer membrane systems by resonance excitation energy transfer, Biochemistry 24: 4097–4108.CrossRefGoogle Scholar
  68. 68.
    Wolf, D. E., Winiski, A. P., Ting, A. E., Bocian, K. M., and Pagano, R. E., 1992, Determination of the transbilayer distribution of fluorescent lipid analogues by nonradiative fluorescence resonance energy transfer, Biochemistry 31: 2865–2873.CrossRefGoogle Scholar
  69. 69.
    Isaacs, B. S., Husten, E. J., Esmon, C. T., and Johnson, A. E., 1986, A domain of membrane-bound blood coagulation factor Va is located far from the phospholipid surface. A fluorescence energy transfer measurement, Biochemistry 25: 4958–4969.CrossRefGoogle Scholar
  70. 70.
    Ladokhin, A. S., Wimley, W. C., Hristova, K., and White, S. H., 1997, Mechanism of leakage of contents of membrane vesicles determined by fluorescence requenching, Methods Enzymol. 278: 474–486.CrossRefGoogle Scholar
  71. 71.
    Kok, J. W., and Hoekstra, D., 1993, Fluorescent lipid analogues applications in cell and membrane biology, Fluorescent and Luminescent Probes for Biological Activity, W. T. Mason (ed.), Academic Press, New York, pp. 101–119.Google Scholar
  72. 72.
    Pyror, C., Bridge, M., and Loew, L. M., 1985, Aggregation, lipid exchange, and metastable phases of dimyristoylphosphatidylethano-lamine vesicles, Biochemistry 24: 2203–2209.CrossRefGoogle Scholar
  73. 73.
    Duzgunes, N., and Bentz, J., 1988, in Spectroscopic Membrane Probes, L. D. Loew (ed.), CRC Press, Boca Raton, Florida, pp. 117–159.Google Scholar
  74. 74.
    Silvius, J. R., and Zuckermann, M. J., 1993, Interbilayer transfer of phospholipid-anchored macromolecules via monomer diffusion, Biochemistry 32: 3153–3161.CrossRefGoogle Scholar
  75. 75.
    Bennett, R. G., 1964, Radiationless intermolecular energy transfer. I. Singlet-singlet transfer, J. Chem. Phys. 41: 3037–3041.CrossRefGoogle Scholar
  76. 76.
    Eisenthal, K. B., and Siegel, S., 1964, Influence of resonance transfer on luminescence decay, J. Chem. Phys. 41: 652–655.CrossRefGoogle Scholar
  77. 77.
    Birks, J. B., and Georghiou, S., 1968, Energy transfer in organic systems VII. Effect of diffusion on fluorescence decay, Proc. R. Soc. (J. Phys. B) 1: 958–965.Google Scholar
  78. 78.
    Thomas, D. D., Caslsen, W. F., and Stryer, L., 1978, Fluorescence energy transfer in the rapid diffusion limit, Proc. Natl. Acad. Sci. U.S.A. 75: 5746–5750.CrossRefGoogle Scholar
  79. 79.
    Selvin, P. R., Rana, T. M., and Hearst, J. E., 1994, Luminescence resonance energy transfer, J. Am. Chem. Soc. 116: 6029–6030.CrossRefGoogle Scholar
  80. 80.
    Li, M., and Selvin, P. R., 1995, Luminescent polyaminocarboxylate chelates of terbium and europium: The effects of chelate structure, J. Am. Chem. Soc. 117: 8132–8138.CrossRefGoogle Scholar
  81. 81.
    Berlman, I. B., 1973, Energy Transfer Parameters of Aromatic Compounds, Academic Press, New York.Google Scholar
  82. 82.
    Fairclough, R. H., and Cantor, C. R., 1978, The use of singlet energy transfer to study macromolecular assemblies, Methods Enzymol. 48: 347–379.CrossRefGoogle Scholar
  83. 83.
    Mathis, G., 1993, Rare earth cryptates and homogeneous fluoroim-munoassays with human sera, Clin. Chem. 39: 1953–1959.Google Scholar
  84. 84.
    Lakowicz, J. R., Johnson, M. L., Wiczk, W., Bhat, A., and Steiner, R. F., 1987, Resolution of a distribution of distances by fluorescence energy transfer and frequency-domain fluorometry, Chem. Phys. Lett. 138: 587–593.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 1999

Authors and Affiliations

  • Joseph R. Lakowicz
    • 1
  1. 1.University of Maryland School of MedicineBaltimoreUSA

Personalised recommendations