Skip to main content
NCBI home page
Biophysical Journal logoLink to Biophysical Journal
. 1988 Aug;54(2):277–288. doi: 10.1016/S0006-3495(88)82957-6

Measurement of protein rotational motion using frequency domain polarized fluorescence depletion.

T M Yoshida 1, F Zarrin 1, B G Barisas 1
PMCID: PMC1330294  PMID: 3207826

Abstract

Polarized fluorescence depletion (PFD) methods (Yoshida, T. M. and B. G. Barisas. Biophys. J. 1986. 50:41-53) are approximately 10(3)-10(4) fold more sensitive than other techniques for measuring protein rotational motions in cell membranes and other viscous environments. Proteins labeled with fluorophores having a high quantum yield for triplet formation are examined anaerobically in a fluorescence microscope. In time domain PFD experiments a several-microsecond pulse of linearly polarized light produces an orientationally-asymmetric depletion of ground state fluorescence in the sample. Monitoring the decay of ground state depletion with a probe beam alternatively polarized, parallel, and perpendicular to the depletion pulse permits the triplet lifetime and rotational correlation time to be resolved and evaluated. We have now explored fluorescence depletion methods in the frequency domain to see whether such measurements could provide simpler and more efficient routine measurements of protein rotational relaxation than previous time domain PFD methods. An acousto-optic modulator (AOM) modulates the intensity of a 514.5 nm argon ion laser beam and a Pockels cell (PC) rotates its plane of polarization. These devices are driven by sinusoidal or square waves in fixed frequency relation, and rigidly phase locked, one to another. The fluorescence emitted from a sample then contains various overtones and combinations of the AOM and PC frequencies. The magnitude and phase of individual fluorescence signal frequencies are measured by a lock-in amplifier using a reference also phase-locked to both the AOM and PC. Specific frequencies permit evaluation of the rotational correlation time of the macromolecule and of the fluorophore triplet state lifetime, respectively. Measurement of bovine serum albumin rotation in glycerol solutions by this method is described.

Full text

PDF
277

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Austin R. H., Chan S. S., Jovin T. M. Rotational diffusion of cell surface components by time-resolved phosphorescence anisotropy. Proc Natl Acad Sci U S A. 1979 Nov;76(11):5650–5654. doi: 10.1073/pnas.76.11.5650. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Barisas B. G., Gill S. J. Thermodynamic analysis of carbon monoxide binding by hemoglobin trout I. Biophys Chem. 1979 Mar;9(3):235–244. doi: 10.1016/0301-4622(79)85006-1. [DOI] [PubMed] [Google Scholar]
  3. Cherry R. J., Cogoli A., Oppliger M., Schneider G., Semenza G. A spectroscopic technique for measuring slow rotational diffusion of macromolecules. 1: Preparation and properties of a triplet probe. Biochemistry. 1976 Aug 24;15(17):3653–3656. doi: 10.1021/bi00662a001. [DOI] [PubMed] [Google Scholar]
  4. Cherry R. J. Rotational and lateral diffusion of membrane proteins. Biochim Biophys Acta. 1979 Dec 20;559(4):289–327. doi: 10.1016/0304-4157(79)90009-1. [DOI] [PubMed] [Google Scholar]
  5. Cherry R. J., Schneider G. A spectroscopic technique for measuring slow rotational diffusion of macromolecules. 2: Determination of rotational correlation times of proteins in solution. Biochemistry. 1976 Aug 24;15(17):3657–3661. doi: 10.1021/bi00662a002. [DOI] [PubMed] [Google Scholar]
  6. Chong P. L., Cossins A. R., Weber G. A differential polarized phase fluorometric study of the effects of high hydrostatic pressure upon the fluidity of cellular membranes. Biochemistry. 1983 Jan 18;22(2):409–415. doi: 10.1021/bi00271a026. [DOI] [PubMed] [Google Scholar]
  7. Garland P. B., Moore C. H. Phosphorescence of protein-bound eosin and erythrosin. A possible probe for measurements of slow rotational mobility. Biochem J. 1979 Dec 1;183(3):561–572. doi: 10.1042/bj1830561. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gratton E., Jameson D. M., Hall R. D. Multifrequency phase and modulation fluorometry. Annu Rev Biophys Bioeng. 1984;13:105–124. doi: 10.1146/annurev.bb.13.060184.000541. [DOI] [PubMed] [Google Scholar]
  9. Johnson P., Garland P. B. Fluorescent triplet probes for measuring the rotational diffusion of membrane proteins. Biochem J. 1982 Apr 1;203(1):313–321. doi: 10.1042/bj2030313. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Keating-Nakamoto S., Cherek H., Lakowicz J. R. A new method for resolution of two- and three-component mixtures of fluorophores by phase-sensitive detection of fluorescence. Anal Biochem. 1985 Aug 1;148(2):349–356. doi: 10.1016/0003-2697(85)90239-8. [DOI] [PubMed] [Google Scholar]
  11. Lakowicz J. R., Cherek H., Maliwal B. P. Time-resolved fluorescence anisotropies of diphenylhexatriene and perylene in solvents and lipid bilayers obtained from multifrequency phase-modulation fluorometry. Biochemistry. 1985 Jan 15;24(2):376–383. doi: 10.1021/bi00323a021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Lakowicz J. R., Gratton E., Cherek H., Maliwal B. P., Laczko G. Determination of time-resolved fluorescence emission spectra and anisotropies of a fluorophore-protein complex using frequency-domain phase-modulation fluorometry. J Biol Chem. 1984 Sep 10;259(17):10967–10972. [PubMed] [Google Scholar]
  13. Lakowicz J. R., Maliwal B. P. Construction and performance of a variable-frequency phase-modulation fluorometer. Biophys Chem. 1985 Jan;21(1):61–78. doi: 10.1016/0301-4622(85)85007-9. [DOI] [PubMed] [Google Scholar]
  14. Lakowicz J. R., Prendergast F. G. Detection of hindered rotations of 1,6-diphenyl-1,3,5-hexatriene in lipid bilayers by differential polarized phase fluorometry. Biophys J. 1978 Oct;24(1):213–231. doi: 10.1016/S0006-3495(78)85357-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Lakowicz J. R., Prendergast F. G., Hogen D. Differential polarized phase fluorometric investigations of diphenylhexatriene in lipid bilayers. Quantitation of hindered depolarizing rotations. Biochemistry. 1979 Feb 6;18(3):508–519. doi: 10.1021/bi00570a021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Lakowicz J. R., Thompson R. B. Differential polarized phase fluorometric studies of phospholipid bilayers under high hydrostatic pressure. Biochim Biophys Acta. 1983 Jul 27;732(2):359–371. doi: 10.1016/0005-2736(83)90052-4. [DOI] [PubMed] [Google Scholar]
  17. Moore C., Boxer D., Garland P. Phosphorescence depolarization and the measurement of rotational motion of proteins in membranes. FEBS Lett. 1979 Dec 1;108(1):161–166. doi: 10.1016/0014-5793(79)81200-4. [DOI] [PubMed] [Google Scholar]
  18. Niswender G. D., Roess D. A., Sawyer H. R., Silvia W. J., Barisas B. G. Differences in the lateral mobility of receptors for luteinizing hormone (LH) in the luteal cell plasma membrane when occupied by ovine LH versus human chorionic gonadotropin. Endocrinology. 1985 Jan;116(1):164–169. doi: 10.1210/endo-116-1-164. [DOI] [PubMed] [Google Scholar]
  19. Squire P. G., Moser P., O'Konski C. T. The hydrodynamic properties of bovine serum albumin monomer and dimer. Biochemistry. 1968 Dec;7(12):4261–4272. doi: 10.1021/bi00852a018. [DOI] [PubMed] [Google Scholar]
  20. Weber G., Helgerson S. L., Cramer W. A., Mitchell G. W. Changes in rotational motion of a cell-bound fluorophore caused by colicin E1: a study by fluorescence polarization and differential polarized phase fluorometry. Biochemistry. 1976 Oct 5;15(20):4429–4432. doi: 10.1021/bi00665a014. [DOI] [PubMed] [Google Scholar]
  21. Yoshida T. M., Barisas B. G. Protein rotational motion in solution measured by polarized fluorescence depletion. Biophys J. 1986 Jul;50(1):41–53. doi: 10.1016/S0006-3495(86)83437-3. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Biophysical Journal are provided here courtesy of The Biophysical Society

RESOURCES