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Biophysical Journal 74: 11-21 (1998)
© 1998 the Biophysical Society

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Biophys J, January 1998, p. 11-21, Vol. 74, No. 1

Donor-Donor Energy Migration for Determining Intramolecular Distances in Proteins: I. Application of a Model to the Latent Plasminogen Activator Inhibitor-1 (PAI-1)

Jan Karolin,* Ming Fa,# Malgorzata Wilczynska,# Tor Ny,# and Lennart B.-Å. Johansson*

 *Department of Physical Chemistry and  #Department of Medical Biochemistry and Biophysics, Umeå University, S-901 87 Umeå, Sweden

A new fluorescence spectroscopic method is presented for determining intramolecular and intermolecular distances in proteins and protein complexes, respectively. The method circumvents the general problem of achieving specific labeling with two different chromophoric molecules, as needed for the conventional donor-acceptor transfer experiments. For this, mutant forms of proteins that contain one or two unique cysteine residues can be constructed for specific labeling with one or two identical fluorescent probes, so-called donors (d). Fluorescence depolarization experiments on double-labeled Cys mutant monitor both reorientational motions of the d molecules, as well as the rate of intramolecular energy migration. In this report a model that accounts for these contributions to the fluorescence anisotropy is presented and experimentally tested. Mutants of a protease inhibitor, plasminogen activator inhibitor type-1 (PAI-1), containing one or two cysteine residues, were labeled with sulfhydryl specific derivatives of 4,4-difluoro-4-borata-3a-azonia-4a-aza-s-indacence (BODIPY). From the rate of energy migration, the intramolecular distance between the d groups was calculated by using the Förster mechanism and by accounting for the influence of local anisotropic orientation of the d molecules. The calculated intramolecular distances were compared with those obtained from the crystal structure of PAI-1 in its latent form. To test the stability of parameters extracted from experiments, synthetic data were generated and reanalyzed.

Biophys J, January 1998, p. 11-21, Vol. 74, No. 1
© 1998 by the Biophysical Society   0006-3495/98/01/11/11  $2.00


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F. Bergstrom, P. Hagglof, J. Karolin, T. Ny, and L. B.-A. Johansson
The use of site-directed fluorophore labeling and donor-donor energy migration to investigate solution structure and dynamics in proteins
PNAS, October 26, 1999; 96(22): 12477 - 12481.
[Abstract] [Full Text] [PDF]


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Copyright © 1998 by the Biophysical Society.