help button home button Biophys. J.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Gautier, I.
Right arrow Articles by Coppey-Moisan, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Gautier, I.
Right arrow Articles by Coppey-Moisan, M.

Biophys J, June 2001, p. 3000-3008, Vol. 80, No. 6

Homo-FRET Microscopy in Living Cells to Measure Monomer-Dimer Transition of GFP-Tagged Proteins

I. Gautier,* M. Tramier,* C. Durieux,* J. Coppey,* R. B. Pansu,dagger J-C. Nicolas,Dagger K. Kemnitz,§ and M. Coppey-Moisan*

 *Institut Jacques Monod, Centre National de la Recherche Scientifique, Université P6/P7, 75251 Paris, France;  dagger Laboratoire de Photophysique et Photochimie Macromoléculaires et Supramoléculaires, 94235 Cachan, France;  Dagger Service de Microbiologie, Hôpital Rothschild, 75571 Paris, France; and  §EuroPhoton GmbH, D-12247, Berlin, Germany

Fluorescence anisotropy decay microscopy was used to determine, in individual living cells, the spatial monomer-dimer distribution of proteins, as exemplified by herpes simplex virus thymidine kinase (TK) fused to green fluorescent protein (GFP). Accordingly, the fluorescence anisotropy dynamics of two fusion proteins (TK27GFP and TK366GFP) was recorded in the confocal mode by ultra-sensitive time-correlated single-photon counting. This provided a measurement of the rotational time of these proteins, which, by comparing with GFP, allowed the determination of their oligomeric state in both the cytoplasm and the nucleus. It also revealed energy homo-transfer within aggregates that TK366GFP progressively formed. Using a symmetric dimer model, structural parameters were estimated; the mutual orientation of the transition dipoles of the two GFP chromophores, calculated from the residual anisotropy, was 44.6 ± 1.6°, and the upper intermolecular limit between the two fluorescent tags, calculated from the energy transfer rate, was 70 Å. Acquisition of the fluorescence steady-state intensity, lifetime, and anisotropy decay in the same cells, at different times after transfection, indicated that TK366GFP was initially in a monomeric state and then formed dimers that grew into aggregates. Picosecond time-resolved fluorescence anisotropy microscopy opens a promising avenue for obtaining structural information on proteins in individual living cells, even when expression levels are very low.

Biophys J, June 2001, p. 3000-3008, Vol. 80, No. 6
© 2001 by the Biophysical Society   0006-3495/01/06/3000/09  $2.00


This article has been cited by other articles:


Home page
 Biophys. JHome page
J. M. Johnson and W. J. Betz
The Color of Lactotroph Secretory Granules Stained with FM1-43 Depends on Dye Concentration
Biophys. J., April 15, 2008; 94(8): 3167 - 3177.
[Abstract] [Full Text] [PDF]

Home page
 Biophys. JHome page
M. A. Digman, R. Dalal, A. F. Horwitz, and E. Gratton
Mapping the Number of Molecules and Brightness in the Laser Scanning Microscope
Biophys. J., March 15, 2008; 94(6): 2320 - 2332.
[Abstract] [Full Text] [PDF]

Home page
 J. Bacteriol.Home page
M. C. Konopka, I. A. Shkel, S. Cayley, M. T. Record, and J. C. Weisshaar
Crowding and confinement effects on protein diffusion in vivo.
J. Bacteriol., September 1, 2006; 188(17): 6115 - 6123.
[Abstract] [Full Text] [PDF]

Home page
 Sci SignalHome page
S. S. Vogel, C. Thaler, and S. V. Koushik
Fanciful FRET
Sci. Signal., April 18, 2006; 2006(331): re2 - re2.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Biol. CellHome page
N. Angelier, M. Tramier, E. Louvet, M. Coppey-Moisan, T. M. Savino, J. R. De Mey, and D. Hernandez-Verdun
Tracking the Interactions of rRNA Processing Proteins during Nucleolar Assembly in Living Cells
Mol. Biol. Cell, June 1, 2005; 16(6): 2862 - 2871.
[Abstract] [Full Text] [PDF]

Home page
 Biophys. JHome page
A. L. Mattheyses, A. D. Hoppe, and D. Axelrod
Polarized Fluorescence Resonance Energy Transfer Microscopy
Biophys. J., October 1, 2004; 87(4): 2787 - 2797.
[Abstract] [Full Text] [PDF]

Home page
 Biophys. JHome page
S. T. Hess, E. D. Sheets, A. Wagenknecht-Wiesner, and A. A. Heikal
Quantitative Analysis of the Fluorescence Properties of Intrinsically Fluorescent Proteins in Living Cells
Biophys. J., October 1, 2003; 85(4): 2566 - 2580.
[Abstract] [Full Text] [PDF]

Home page
 Biophys. JHome page
J. V. Rocheleau, M. Edidin, and D. W. Piston
Intrasequence GFP in Class I MHC Molecules, a Rigid Probe for Fluorescence Anisotropy Measurements of the Membrane Environment
Biophys. J., June 1, 2003; 84(6): 4078 - 4086.
[Abstract] [Full Text] [PDF]

Home page
 Biophys. JHome page
M. Tramier, I. Gautier, T. Piolot, S. Ravalet, K. Kemnitz, J. Coppey, C. Durieux, V. Mignotte, and M. Coppey-Moisan
Picosecond-Hetero-FRET Microscopy to Probe Protein-Protein Interactions in Live Cells
Biophys. J., December 1, 2002; 83(6): 3570 - 3577.
[Abstract] [Full Text] [PDF]

Home page
 Biophys. JHome page
A. H. A. Clayton, Q. S. Hanley, D. J. Arndt-Jovin, V. Subramaniam, and T. M. Jovin
Dynamic Fluorescence Anisotropy Imaging Microscopy in the Frequency Domain (rFLIM)
Biophys. J., September 1, 2002; 83(3): 1631 - 1649.
[Abstract] [Full Text] [PDF]


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Copyright © 2001 by the Biophysical Society.