Application of donor-donor energy migration (DDEM) for examining protein structure and function

Fredrik Bergstrom; Peter Hagglof; Jan Karolin; Tor Ny; Lennart B.-A. Johansson

doi:10.1117/12.347528

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3 May 1999 Application of donor-donor energy migration (DDEM) for examining protein structure and function

Fredrik Bergstrom, Peter Hagglof, Jan Karolin, Tor Ny, Lennart B.-A. Johansson

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Proceedings Volume 3602, Advances in Fluorescence Sensing Technology IV; (1999) https://doi.org/10.1117/12.347528
Event: BiOS '99 International Biomedical Optics Symposium, 1999, San Jose, CA, United States

Abstract

Donor-Donor Energy Migration (DDEM) and fluorescence anisotropy experiments can be utilized as a versatile tool for examining protein structure and function. For this, pairs of identical fluorescent probes (D) are attached to unique residues created by means of site specific mutagenesis. Present work illustrates the applicability of the method on the latent form of Plasminogen Activator Inhibitor-1 (PAI-1). Different DD-pairs of mutated PAI-1 were prepared and studied, namely; V106C-H185C, H185C-M266C and M266C-V106C. The Cys residues were labelled with a sulfhydryl specific derivative of BODIPY^R [N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a- diaza-s-indacene-3-yl)methyl iodo-acetamide]. To determine the rate of DDEM within such a pair, intramolecular order and dynamics must be considered (Biophys. J., 74, 11-21, 1997). For analysis of data, additonal information was obtained from experiments with the corresponding D-labelled single Cys mutants, that is, V106C, H185C and M266C. The stability of values determined was tested by generating and re-analyzing synthetic data. The intramolecular distances obtained agree, reasonably well, with those determined from the X-ray structure of latent PAI-1.

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Fredrik Bergstrom, Peter Hagglof, Jan Karolin, Tor Ny, and Lennart B.-A. Johansson "Application of donor-donor energy migration (DDEM) for examining protein structure and function", Proc. SPIE 3602, Advances in Fluorescence Sensing Technology IV, (3 May 1999); https://doi.org/10.1117/12.347528

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