Rotational Diffusion of Membrane Proteins Optical Methods

  • Peter B. Garland
  • Pauline Johnson


Optical methods for measurement of Brownian rotational diffusion depend upon the use of linearly polarized light. Furthermore, the molecule under study must be capable of being photoexcited to some state which can be detected separately from the non-excited state. The measurements of rotation depend upon photoselection, which briefly works as follows. An isotropic (random) array of molecules is partially converted to the excited state by a flash of polarized light of appropriate wavelength. Because of the relationship between molecular orientation and the probability of absorbing linearly polarized light, the population of excited molecules is anisotropic. This anisotropy can be detected optically in various ways: by polarization of light emitted from the excited state, e.g., prompt and delayed fluorescence and phosphorescence, by polarized absorption measurements of the excited state absorption bands, i.e., linear dichroism, or of the remaining and also anisotropic ground-state absorption bands. Rotational diffusion will abolish the flash-established anisotropy in a time-dependent fashion. Conversely, measurements of the decay of flash-induced anisotropy enable rotational diffusion coefficients to be calculated. These concepts were established more than half a century ago (Perrin, 1926, 1929) and have been extensively reviewed (Albrecht, 1961, 1970).


Rotational Diffusion Transition Dipole Moment Delayed Fluorescence Linear Dichroism Rotational Mobility 


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  1. Albrecht, A. C., 1961, Polarisations and assignments of transitions: The method of photoselection, J. Mol. Spectrosc. 6:301–334.CrossRefGoogle Scholar
  2. Albrecht, A. C., 1970, The method of photoselection and some recent applications, Prog. React. Kinet. 5:301–334.Google Scholar
  3. Austin, R. H., Chan, S. S., and Jovin, T. M., 1979, Rotational diffusion of cell surface components by time-resolved phosphorescence anisotropy, Proc. Natl. Acad. Sci. USA 76:5650–5654.PubMedCrossRefGoogle Scholar
  4. Axelrod, D., Koppel, D. E., Schlessinger, J., Elson, E., and Webb, W. W., 1976, Mobility measurement by analysis of fluorescence photobleaching recovery kinetics, Biophys. J. 16:1055–1069.PubMedCrossRefGoogle Scholar
  5. Bartholdi, M., Barrantes, F. J., and Jovin, T. M., 1981, Rotational molecular dynamics of the membrane-bound acetylcholine receptor revealed by phosphorescence spectroscopy, Eur. J. Biochem. 120:389–396.PubMedCrossRefGoogle Scholar
  6. Belford, G. G., Belford, R. L., and Weber, G., 1972, Dynamics of fluorescence polarizations in macromolecules, Proc. Natl. Acad. Sci. USA 69:1392–1393.PubMedCrossRefGoogle Scholar
  7. Brand, L., and Witholt, B., 1967, Fluorescence measurements, Meth. Enzymol. 11:776–857.CrossRefGoogle Scholar
  8. Burkli, A., and Cherry, R. J., 1981, Rotational motion and flexibility of Ca2+ and Mg2+ dependent adenosine 5′-triphosphatase in sarcoplasmic reticulum membranes, Biochemistry 20:138–145.PubMedCrossRefGoogle Scholar
  9. Chapman, D., and Restall, C. J., 1982, Rotational and lateral movements in biomembranes: The dynamics of biomembrane components, Biochem. Soc. Symp. 46:139–154.Google Scholar
  10. Cherry, R. J., 1978, Measurement of protein rotational diffusion in membranes by flash photolysis, Meth. Enzymol. 54:47–61.PubMedCrossRefGoogle Scholar
  11. Cherry, R. J., 1979, Rotational and lateral diffusion of membrane proteins, Biochim. Biophys. Acta 559:289–327.PubMedCrossRefGoogle Scholar
  12. Cherry, R. J., and Godfrey, R. E., 1981, Anisotropie rotation of bacteriorhodopsin in lipid membranes. Comparison of theory with experiment, Biophys. J. 36:251–216.CrossRefGoogle Scholar
  13. Cherry, R. J., and Schneider, G., 1976, A spectroscopic technique for measuring slow rotational diffusion of macromolecules. 2: Determination of rotational correlation times of proteins in solution, Biochemistry 15:3657–3661.PubMedCrossRefGoogle Scholar
  14. Cherry, R. J., Cogoli, A., Oppliger, M., Schneider, G., and Semenza, G., 1976a, A spectroscopic technique for measuring slow rotational diffusion of macromolecules. I: Preparation and properties of a triplet probe, Biochemistry 15:3653–3656.PubMedCrossRefGoogle Scholar
  15. Cherry, R. J., Burkli, A., Busslinger, M., Schneider, G., and Parish, G. R., 1976b, Rotational diffusion of band 3 proteins in the human erythrocyte membrane, Nature (London) 263:389–393.CrossRefGoogle Scholar
  16. Cherry, R. J., Müller, U., and Schneider, G., 1977, Rotational diffusion of bacteriorhodopsin in lipid membranes, FEBS Lett. 80:465–469.PubMedCrossRefGoogle Scholar
  17. Chuang, T. J., and Eisenthal, K. B., 1972, Theory of fluorescence depolarization by anisotropic rotational diffusion, J. Chem. Phys. 57:5094–5097.CrossRefGoogle Scholar
  18. Cone, R. A., 1972, Rotational diffusion of rhodopsin in the visual receptor membrane, Nature (London) New Biol. 236:39–43.Google Scholar
  19. Dixit, R. P. S., Waring, A. J., Wells, K. O., Wong, P. S., Woodrow, G. V., and Vanderkooi, J. M., 1982, Rotational motion of cytochrome c derivatives bound to membranes measured by fluorescence and phosphorescence anisotropy, Eur. J. Biochem. 126:1–9.PubMedCrossRefGoogle Scholar
  20. Ehrenberg, M., and Rigler, R., 1976, Fluorescence correlation spectroscopy applied to rotational diffusion of macromolecules, Quart. Rev. Biophys. 9:69–81.CrossRefGoogle Scholar
  21. Erecinska, M., Wilson, D. F., and Blasie, J. K., 1978, Studies on the orientation of the mitochondrial redox carriers. I. Orientation of the hemes of cytochrome c oxidase with respect to the plane of a cytochrome oxidase lipid model membrane, Biochim. Biophys. Acta 501:53–62.PubMedCrossRefGoogle Scholar
  22. Frye, L. D., and Edidin, M., 1970, The rapid intermixing of cell surface antigens after formation, J. Cell Sci. 7:319–335.PubMedGoogle Scholar
  23. Garland, P., 1981, Letter to the editor. Fluorescence photobleaching recovery: Control of laser intensities with an acousto-optic modulator, Biophys. J. 33:481–482.PubMedCrossRefGoogle Scholar
  24. Garland, P. B., and Johnson, P., 1983, Rotational diffusion measured by depolarization of fluorescence depletion, in: Spectroscopy and the Dynamics of Biological Systems (P. Bailey and R. E. Dale), in press.Google Scholar
  25. Garland, P. B., and Moore, C. H., 1979, Phosphorescence of protein-bound eosin and erythrosin. A possible probe for measurements of slow rotational mobility, Biochem. J. 183:561–572.PubMedGoogle Scholar
  26. Garland, P. B., Davison, M. T., and Moore, C. H., 1979, Rotational mobility of membrane-bound cytochrome o of Escherichia coli and cytochrome a1 of Thiobacillus ferro-oxidans, Biochem. Soc. Trans. 7:1112–1113.PubMedGoogle Scholar
  27. Gouterman, M., and Stryer, L., 1962, Fluorescence polarization of some porphyrins, J. Chem. Phys. 37:2260–2266.CrossRefGoogle Scholar
  28. Greinhert, R., Staerk, H., Stier, A., and Weiler, A., 1979, E-type delayed fluorescence depolarization, a technique to probe rotational motion in the microsecond range, J. Biochem. Biophys. Meth. 1:77–83.CrossRefGoogle Scholar
  29. Gut, J., Richter, C., Cherry, R. J., Winterhalter, K. H., and Kawato, S., 1982, Rotation of cytochrome P-450. II. Specific interactions of cytochrome P-450 with NADPH-cytochrome P-450 reductase in phospholipid vesicles, J. Biol. Chem. 257:7030–7036.PubMedGoogle Scholar
  30. Gut, J., Richter, C., Cherry, R. J., Winterhalter, K. H., and Kawato, S., 1983, Rotation of cytochrome P-450. Complex formation of cytochrome P-450 with NADPH-cytochrome P-450 reductase in lipo-somes, J. Biol. Chem. 258:8588–8594.PubMedGoogle Scholar
  31. Henderson, R., and Unwin, P. N. T., (1975), Three-dimensional model of purple membrane obtained by electron microscopy, Nature (London) 257:28–32.CrossRefGoogle Scholar
  32. Heyn, M. P., Cherry, R. J., and Dencher, N. A., 1981, Lipid-protein interactions in bacteriorhodopsin-dimyristoylphosphatidylcholine vesicles, Biochemistry 20:840–849.PubMedCrossRefGoogle Scholar
  33. Hoffman, W., Sarzala, M. G., and Chapman, D., 1979, Rotational motion and evidence for oligomeric structures of sarcoplasmic reticulum Ca2+-activated ATPase, Proc. Natl. Acad. Sci. USA 76:3860–3864.CrossRefGoogle Scholar
  34. Hoffman, W., Sarzala, M. G., Gomezfernandez, J., Goni, F. M., Restall, C. J., Chapman, D., 1980, Protein rotational diffusion and lipid structure of reconstituted systems of Ca2+-activated adenosine triphosphatase, J. Mol. Biol. 141:119–132.CrossRefGoogle Scholar
  35. Inbar, M., Shinitzky, M., and Sachs, L., 1973, Rotational relaxation time of concanavalin A bound to the surface membrane of normal and malignant transformed cells, J. Mol. Biol. 81:245–253.PubMedCrossRefGoogle Scholar
  36. Jablonski, V. A., 1961, Uber die abklingungsvorgange polarisierter photolunineszenz, Aeitschrift Natur-forsch. 16:1–4.Google Scholar
  37. Johnson, P., 1983, The lateral and rotational mobility of membrane components measured by fluorescence recovery after photobleaching and fluorescence depletion recovery, Ph.D. thesis, University of Dundee.Google Scholar
  38. Johnson, P., and Garland, P. B., 1981, Depolarization of fluorescence depletion: A microscopic method for measuring rotational diffusion of membrane proteins on the surface of a single cell, FEBS Lett. 132:252–256.PubMedCrossRefGoogle Scholar
  39. Johnson, P., and Garland, P. B., 1982a, Fluorescent triplet probes for measuring the rotational diffusion of membrane proteins, Biochem. J. 203:313–321.PubMedGoogle Scholar
  40. Johnson, P., and Garland, P. B., 1982b, Carbocyanine dyes used as fluorescent triplet probes for measuring slow rotational diffusion of lipids in membranes, Biochem. J. 203:313–394.PubMedGoogle Scholar
  41. Jovin, T. M., Bartholdi, M., Vaz, W. L. C., and Austin, R. H., 1981, Rotational diffusion of biological macromolecules by time-resolved delayed luminescence (phosphorescence, fluorescence) anisotropy, Ann. N.Y. Acad. Sci. USA 399:176–196.CrossRefGoogle Scholar
  42. Junge, W., 1972, Brownian rotation of the cytochrome oxidase in the mitochondrial membrane, FEBS Lett. 25:109–112.PubMedCrossRefGoogle Scholar
  43. Junge, W., and Devault, D., 1975, Symmetry, orientation and rotational mobility in the a3 heme of cytochrome c oxidase in the inner membrane of mitochondria, Biochem. Biophys. Acta 408:200–214.PubMedCrossRefGoogle Scholar
  44. Kawato, S., and Kinosita, K., 1981, Time dependent absorption anisotropy and rotational diffusion of proteins in membranes, Biochem. J. 36:227–296.Google Scholar
  45. Kawato, S., Sigel, E., Carafoli, E., and Cherry, R. J., 1980, Cytochrome oxidase rotates in the inner membrane of intact mitochondria and submitochondrial particles, J. Biol. Chem. 255:5508–5510.PubMedGoogle Scholar
  46. Kawato, S., Sigel, E., Carafoli, E., and Cherry, R. J., 1981, Rotation of cytochrome oxidase in phospholipid vesicles. Investigations of interactions between cytochrome oxidases and between cytochrome oxidase and cytochrome bc 1 complex, J. Biol. Chem. 256:7518–7527.PubMedGoogle Scholar
  47. Kawato, S., Gut, J., Cherry, R. J., Winterhalter, K. H., and Richter, C., 1982a, Rotation of cytochrome P-450. I. Investigation of protein—protein interactions of cytochrome P-450 in phospholipid vesicles and liver microsomes, J. Biol. Chem. 257:7023–7029.PubMedGoogle Scholar
  48. Kawato, S., Lehner, C., Müller, M., and Cherry, R. J., 1982b, Protein-protein interactions of cytochrome oxidase in inner mitochondrial membranes. The effect of liposome fusion on protein rotational mobility, J. Biol. Chem. 257:6470–6476.PubMedGoogle Scholar
  49. Koppel, D. E., Axelrod, D., Schlessinger, J., Elson, E. L., and Webb, W. W., 1976, Dynamics of fluorescence marker concentration as a probe of mobility, Biophys. J. 16:1315–1329.PubMedCrossRefGoogle Scholar
  50. Kunze, U., and Junge, W., 1977, Ellipticity of cytochrome a 3 and rotational mobility of cytochrome c-oxidase in the cristae membrane of mitochondria, FEBS Lett. 80:429–434.PubMedCrossRefGoogle Scholar
  51. Lakowicz, J. R., Prendergast, F. G., and Hogan, D., 1979, Differential polarized phase fluorometric investigations of diphenylhexatriene in lipid bilayers. Quantitation of hindered depolarizing rotations, Biochemistry 18:508–519.PubMedCrossRefGoogle Scholar
  52. Lavalette, D., and Amand, B., and Pochon, F., 1977, Rotational relaxation of 70S ribosome by a depolarization method using triplet probes, Proc. Natl. Acad. Sci. USA 74:1407–1411.PubMedCrossRefGoogle Scholar
  53. Lindmo, T., and Steen, H. B., 1977, The effect of numerical aperture of detector optics on polarization values, Biophys. J. 18:173–187.PubMedCrossRefGoogle Scholar
  54. Lo, M. M. S., Garland, P. B., Lamprecht, J., and Barnard, E. A., 1980, Rotational mobility of the membrane-bound acetylcholine receptor of Torpedo electric organ measured by phosphorescence depolarization, FEBS Lett. 111:407–412.PubMedCrossRefGoogle Scholar
  55. Mar, T., Picorel, R., and Gingras, G., 1981, Rotational mobility of the photoreaction center in chromophore membranes of Rhodospirillum rubrum, Biochim. Biophys. Acta 637:546–550.CrossRefGoogle Scholar
  56. Matayoshi, E. D., Corin, A. F., Zidovetzki, R., Sawyer, W. H., and Jovin, T. M., 1982, Rotational dynamics of cell surface proteins, in: Proceedings of the FEBS Symposium Konstanz. Mobility and Recognition in Cell Biology. (H. Sund and C. Veeger, eds.), Walter de Gruyter & Co., Berlin.Google Scholar
  57. Moore, C. H., and Garland, P. B., 1979, Synthesis of erythrosin isothiocyanate and its use as a phosphorescence depolarization probe for slow rotational mobility of membrane proteins, Biochem. Soc. Trans. 7:945–946.PubMedGoogle Scholar
  58. Moore, C., Boxer, D., and Garland, P., 1979, Phosphorescence depolarization and the measurement of rotational motion of proteins in membranes, FEBS Lett. 108:161–166.PubMedCrossRefGoogle Scholar
  59. Muller, M., Krebs, J. J. R., Cherry, R. J., and Kawato, S., 1982, Selective labeling and rotational diffusion of the ADP/ATP translocator in the inner mitochondrial membrane, J. Biol. Chem. 257:1117–1120.PubMedGoogle Scholar
  60. Murray, E. K., Restall, C. J., and Chapman, D., 1983, Monitoring membrane protein rotational diffusion using time-averaged phosphorescence, Biochim. Biophys. Acta 732:347–351.PubMedCrossRefGoogle Scholar
  61. Naqvi, K. R., and Wild, U. P., 1975, The use of E-type delayed fluorescence for probing rotational relaxation, Chem. Phys. Lett. 36:222–224.CrossRefGoogle Scholar
  62. Naqvi, K. R., Gonzales, R. J., Cherry, R. J., and Chapman, D., 1973, Spectroscopic technique for studying protein rotation in membranes, Nature (London) New Biol. 245:249–251.CrossRefGoogle Scholar
  63. Nigg, E. A., and Cherry, R. J., 1979, Influence of temperature and cholesterol on the rotational diffusion of band 3 in the human erythrocyte membrane, Biochemistry 18:3457–3465.PubMedCrossRefGoogle Scholar
  64. Nigg, E. A., and Cherry, R. J., 1980, Anchorage of a band 3 population at the erythrocyte cytoplasmic membrane surface: Protein rotational diffusion measurements, Proc. Natl. Acad. Sci. USA 77:4702–4706.PubMedCrossRefGoogle Scholar
  65. Perrin, P. F., 1926, Polarisation de la lumiere de fluorescence. Vie moyenne des molecules dans l’etat excite, J. Phys. Rad. 7:390.CrossRefGoogle Scholar
  66. Perrin, P. F., 1929, La fluorescence des solutions. Induction moleculaire.—polarization et duree d’emission-photochimiew, Annde Phys. 12:169–275.Google Scholar
  67. Peters, R., and Cherry, R. J., 1982, Lateral and rotational diffusion of bacteriorhodopsin in lipid bilayers: An experimental test of the Saffmann—Delbriick equations, Proc. Natl. Acad. Sci. USA 79:4317–4321.PubMedCrossRefGoogle Scholar
  68. Peters, R., Peters, J., Tews, K. H., and Bahr, W., 1974, A microfluorometric study of translational diffusion in erythrocyte membranes, Biochim. Biophys. Acta 367:282–294.PubMedCrossRefGoogle Scholar
  69. Pilipovich, V. A., 1961, Polarization of phosphorescence in organophors, Opt. Spectrosc. 10:104–107.Google Scholar
  70. Richter, C., Winterhalter, K. H., and Cherry, R. J., 1979, Rotational diffusion of cytochrome P-450 in rat liver microsomes, FEBS Lett. 102:151–154.PubMedCrossRefGoogle Scholar
  71. Rigler, R., and Ehrenberg, M., 1973, Molecular interactions and structure as analysed by fluorescence relaxation spectroscopy, Quart. Rev. Biophys. 6:139–199.CrossRefGoogle Scholar
  72. Saffman, R. G., and Delbrück, M., 1975, Brownian motion in biological membranes, Proc. Natl. Acad. Sci. USA 72:3111–3113.PubMedCrossRefGoogle Scholar
  73. Shinitzky, M., Inbar, M., and Sachs, L., 1973, Rotational diffusion of lectins bound to the surface membrane of normal lymphocytes, FEBS Lett. 34:247–250.PubMedCrossRefGoogle Scholar
  74. Singer, S., and Nicholson, G. L., 1972, The final mosaic model of the structure of cell membranes, Science 175:720–731.PubMedCrossRefGoogle Scholar
  75. Smith, L., Weis, R. M., and McConnell, H. M., 1981, Measurement of rotational motion in membranes using fluorescence recovery after photobleaching, Biophys. J. 36:73–91.PubMedCrossRefGoogle Scholar
  76. Spiers, A., Moore, C. H., Boxer, D. H., and Garland, P. B., 1983, Segmental motion and rotational diffusion of the Ca2+-translocating ATPase of sarcoplasmic reticulum, measured by time-resolved phosphorescence depolarization, Biochem. J. 213:67–74.Google Scholar
  77. Strambini, G. B., and Galley, W. C., 1976, Detection of slow rotational motions of proteins by steady-state phosphorescence anisotropy, Nature (London) 260:554–556.CrossRefGoogle Scholar
  78. Strambini, G. B., and Galley, W. C., 1980, Time-dependent phosphorescence anisotropy measurements of the slow rotational motions of proteins in viscous solution, Biopolymers 19:383–394.CrossRefGoogle Scholar
  79. Teale, F. W. J., 1969, Fluorescence depolarization by light-scattering in turbid solutions, Photobiochem. Photobiophys. 10:363–374.CrossRefGoogle Scholar
  80. Trauble, H., and Sackmann, E., 1973, Lipid motion and rhodopsin rotation, Nature (London) 245:210–211.CrossRefGoogle Scholar
  81. Valeur, B., and Weber, G., 1977, Anisotropic rotations in 1-naphthylamine. Existence of a red-edge transition moment normal to the ring plane, Chem. Phys. Lett. 45:140–144.CrossRefGoogle Scholar
  82. Vaz, W. L. C., Austin, R. H., and Vogel, H., 1979, The rotational diffusion of cytochrome b 5 in lipid bilayer membranes. Influence of the lipid physical state, Biophys. J. 26:415–426.PubMedCrossRefGoogle Scholar
  83. Vaz, W. L. C., Criado, M., Madeira, V. M. C., Schoellmann, G., and Jovin, T. M., 1982, Size dependence of the translational diffusion of large integral membrane proteins in liquid-crystalline phase lipid bilayers. A study using FRAP, Biochemistry 21:5608–5612.PubMedCrossRefGoogle Scholar
  84. Wagner, R., and Junge, W., 1982, Coupling factor for photophosphorylation labelled with eosin isothiocyanate: Activity, size and shape in solution, Biochemistry 21:1890–1899.PubMedCrossRefGoogle Scholar
  85. Wagner, R., Carrillo, N., Junge, W., and Vallejos, R. H., 1981, Heat-activated conformational changes of isolated coupling factor of photophosphorylation CF1, FEBS Lett. 136:208–212.CrossRefGoogle Scholar
  86. Wagner, R., Carillo, N., Junge, W., and Vallejos, R. H., 1982, On the conformation of reconstituted ferredoxin: NADP oxidoreductase in the thylakoid membrane. Studies via triplet lifetime and rotational diffusion with eosin isothiocyanate as label, Biochim. Biophys. Acta 680:317–330.CrossRefGoogle Scholar
  87. Wagner, R., Andreo, C., and Junge, W., 1983, Evidence for a sequestered solvent space in the chloroplast ATPase, Biochim. Biophys. Acta 723:123–127.CrossRefGoogle Scholar
  88. Wahl, P., Kasai, M., and Changeux, J. P., 1971, A study on the motion of proteins in excitable membrane fragments by nanosecond fluorescence polarization spectroscopy, Eur. J. Biochem. 36:257–276.Google Scholar
  89. Weber, G., 1953, Rotation, Brownian motion and polarization of the fluorescence of solutions, Adv. Protein Chem. 8:415–459.PubMedCrossRefGoogle Scholar
  90. Weber, G., 1977, Theory of differential phase fluorometry: Detection of anisotropic molecular rotations, J. Chem. Phys. 66:4081–4091.CrossRefGoogle Scholar
  91. Willingham, M. C., and Pastan, A., 1978, The visualization of fluorescent proteins in living cells by video intensification microscopy, Clin. Endocrinol. 13:501–507.Google Scholar
  92. Zidovetzki, R., Yarden, Y., Schlessinger, J., and Jovin, T. M., 1981, Rotational diffusion of epidermal growth factor complexed to cell surface receptors reflects rapid microaggregation and endocytosis of occupied receptors, Proc. Natl. Acad. Sci. USA 78:6981–6985.PubMedCrossRefGoogle Scholar

Copyright information

© Plenum Press, New York 1985

Authors and Affiliations

  • Peter B. Garland
    • 1
  • Pauline Johnson
    • 1
  1. 1.Department of BiochemistryUniversity of DundeeDundeeScotland, UK

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