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The use of site-directed fluorophore labeling and donor-donor energy migration to investigate solution structure and dynamics in proteins.
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
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1999 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 96, no 22, 12477-81 p.Article in journal (Refereed) Published
Abstract [en]

The use of molecular genetics for introducing fluorescent molecules enables the use of donor-donor energy migration to determine intramolecular distances in a variety of proteins. This approach can be applied to examine the overall molecular dimensions of proteins and to investigate structural changes upon interactions with specific target molecules. In this report, the donor-donor energy migration method is demonstrated by experiments with the latent form of plasminogen activator inhibitor type 1. Based on the known x-ray structure of plasminogen activator inhibitor type 1, three positions forming the corners of a triangle were chosen. Double Cys substitution mutants (V106C-H185C, H185C-M266C, and M266C-V106C) and corresponding single substitution mutants (V106C, H185C, and M266C) were created and labeled with a sulfhydryl specific derivative of BODIPY (=the D molecule). The side lengths of this triangle were obtained from analyses of the experimental data. The analyses account for the local anisotropic order and rotational motions of the D molecules, as well as for the influence of a partial DD-labeling. The distances, as determined from x-ray diffraction, between the C(alpha)-atoms of the positions V106C-H185C, H185C-M266C, and M266C-V106C were 60.9, 30.8, and 55.1 A, respectively. These are in good agreement with the distances of 54 +/- 4, 38 +/- 3, and 55 +/- 3 A, as determined between the BODIPY groups attached via linkers to the same residues. Although the positions of the D-molecules and the C(alpha)-atoms physically cannot coincide, there is a reasonable agreement between the methods.

Place, publisher, year, edition, pages
1999. Vol. 96, no 22, 12477-81 p.
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Medical and Health Sciences
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URN: urn:nbn:se:umu:diva-59653PubMedID: 10535947OAI: oai:DiVA.org:umu-59653DiVA: diva2:555727
Available from: 2012-09-21 Created: 2012-09-21 Last updated: 2017-12-07

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