Skip to main content
NCBI home page
Biophysical Journal logoLink to Biophysical Journal
. 1986 Jul;50(1):41–53. doi: 10.1016/S0006-3495(86)83437-3

Protein rotational motion in solution measured by polarized fluorescence depletion.

T M Yoshida, B G Barisas
PMCID: PMC1329657  PMID: 3730506

Abstract

A microscope-based system is described for directly measuring protein rotational motion in viscous environments such as cell membranes by polarized fluorescence depletion (PFD). Proteins labeled with fluorophores having a high quantum yield for triplet formation, such as eosin isothiocyanate (EITC), are examined anaerobically in a fluorescence microscope. An acousto-optic modulator generates a several-microsecond pulse of linearly polarized light which produces an orientationally-asymmetric depletion of ground state fluorescence in the sample. When the sample is then probed with light polarized parallel to the excitation pulse, fluorescence recovers over 0-1,000 microseconds as the sum of two exponentials. One exponential corresponds to triplet decay and the other to the rotational relaxation. An exciting pulse perpendicular to the probe beam is then applied. Fluorescence recovery following this pulse is the difference of the same two exponentials. Equations for fluorescence recovery kinetics to be expected in various experimentally significant cases are derived. Least-squares analysis using these equations then permits the triplet lifetime and rotational correlation time to be determined directly from PFD data. Instrumentation for PFD measurements is discussed that permits photobleaching recovery measurements of lateral diffusion coefficients using the same microscope system. With this apparatus, both rotational and translational diffusion coefficients (Dr, Dt) were measured for EITC-labeled bovine serum albumin in glycerol solutions. Values obtained for Dr and Dt are discussed in light of both the PFD models and the experimental system.

Full text

PDF
41

Selected References

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

  1. Axelrod D. Carbocyanine dye orientation in red cell membrane studied by microscopic fluorescence polarization. Biophys J. 1979 Jun;26(3):557–573. doi: 10.1016/S0006-3495(79)85271-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Axelrod D. Cell surface heating during fluorescence photobleaching recovery experiments. Biophys J. 1977 Apr;18(1):129–131. doi: 10.1016/S0006-3495(77)85601-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Barisas B. G. Criticality of beam alignment in fluorescence photobleaching recovery experiments. Biophys J. 1980 Mar;29(3):545–548. doi: 10.1016/S0006-3495(80)85153-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Barisas B. G., Leuther M. D. Fluorescence photobleaching recovery measurement of protein absolute diffusion constants. Biophys Chem. 1979 Sep;10(2):221–229. doi: 10.1016/0301-4622(79)85044-9. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. 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]
  7. Damjanovich S., Trón L., Szöllösi J., Zidovetzki R., Vaz W. L., Regateiro F., Arndt-Jovin D. J., Jovin T. M. Distribution and mobility of murine histocompatibility H-2Kk antigen in the cytoplasmic membrane. Proc Natl Acad Sci U S A. 1983 Oct;80(19):5985–5989. doi: 10.1073/pnas.80.19.5985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Edelman G. M. Surface modulation in cell recognition and cell growth. Science. 1976 Apr 16;192(4236):218–226. doi: 10.1126/science.769162. [DOI] [PubMed] [Google Scholar]
  9. Johnson P., Garland P. B. Depolarization of fluorescence depletion. A microscopic method for measuring rotational diffusion of membrane proteins on the surface of a single cell. FEBS Lett. 1981 Sep 28;132(2):252–256. doi: 10.1016/0014-5793(81)81172-6. [DOI] [PubMed] [Google Scholar]
  10. Jähnig F. Structural order of lipids and proteins in membranes: evaluation of fluorescence anisotropy data. Proc Natl Acad Sci U S A. 1979 Dec;76(12):6361–6365. doi: 10.1073/pnas.76.12.6361. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kuntz I. D., Jr, Kauzmann W. Hydration of proteins and polypeptides. Adv Protein Chem. 1974;28:239–345. doi: 10.1016/s0065-3233(08)60232-6. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Peacock J. S., Barisas B. G. Photobleaching recovery studies of antigen-specific mouse lymphocyte stimulation by DNP-conjugated polymerized flagellin. J Immunol. 1981 Sep;127(3):900–906. [PubMed] [Google Scholar]
  14. Peters R., Cherry R. J. Lateral and rotational diffusion of bacteriorhodopsin in lipid bilayers: experimental test of the Saffman-Delbrück equations. Proc Natl Acad Sci U S A. 1982 Jul;79(14):4317–4321. doi: 10.1073/pnas.79.14.4317. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Peters R. Translational diffusion in the plasma membrane of single cells as studied by fluorescence microphotolysis. Cell Biol Int Rep. 1981 Aug;5(8):733–760. doi: 10.1016/0309-1651(81)90231-9. [DOI] [PubMed] [Google Scholar]
  16. Saffman P. G., Delbrück M. Brownian motion in biological membranes. Proc Natl Acad Sci U S A. 1975 Aug;72(8):3111–3113. doi: 10.1073/pnas.72.8.3111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Speirs A., Moore C. H., Boxer D. H., Garland P. B. Segmental motion and rotational diffusion of the Ca2+-translocating adenosine triphosphatase of sarcoplasmic reticulum, measured by time-resolved phosphorescence depolarization. Biochem J. 1983 Jul 1;213(1):67–74. doi: 10.1042/bj2130067. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. Wegener W. A. Fluorescence recovery spectroscopy as a probe of slow rotational motions. Biophys J. 1984 Dec;46(6):795–803. doi: 10.1016/S0006-3495(84)84078-3. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES