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- Edman, Peter, et al.
(författare)
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Extended Förster theory of donor-donor energy migration in bifluorophoric macromolecules. Part II : Method for determining intramolecular distances with experimental validation using mono and bifluorophoric systems
- 2000
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Ingår i: Physical chemistry chemical physics. ; 2:12, s. 2795-2801
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Tidskriftsartikel (refereegranskat)abstract
- Recently an approximate theory was presented and applied for determining intramolecular distances in proteins. The rate of donor-donor energy migration (DDEM) is extracted and analysed from fluorescence depolarisation experiments by means of the DDEM model (Karolin et al., Biophys. J., 1998, 74, 11; Bergström et al., Proc. Natl. Acad. Sci., 1999, 96, 12477). Previously an extended Förster theory (EFT) was derived (Johansson et al., J. Chem. Phys., 1996, 105, 10896), which accounts for DDEM between reorienting molecules. For the first time, this rigorous theory is applied for analysing time-resolved fluorescence depolarisation data, accumulated by using the time-correlated single photon counting (TCSPC) technique. A simulation-deconvolution algorithm is presented which reduces the need of the DDEM model (Edman et al., Phys. Chem. Chem. Phys., 2000, 2, 1789), and other approximate theories (Edman et al., Mol. Phys., submitted). Two bifluorophoric systems were studied, namely; 1,32-dihydroxy-dotriacontane-bis(rhodamine) 101 ester solubilised in lipid vesicles, and bis(9-anthrylmethyl-phosphonate) bisteroid dissolved in propane-1,2-diol. The bis-rhodamine molecules span across lipid bilayers, so that the two rhodamine moieties of the molecule are localised on opposite sides of a bilayer. From the analyses of the fluorescence anisotropy, the donor-donor distances were determined to be 36.5 ± 1 and 21.0 ± 1.5 Å, for the membrane spanning molecule and the bisteroid, respectively. The results are in good agreement with independent studies.
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| 3. |
- Fa, Ming, et al.
(författare)
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The structure of a serpin–protease complex revealed by intramolecular distance measurements using donor–donor energy migration and mapping of interaction sites
- 2000
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Ingår i: Structure. ; 8:4, s. 397-405
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Tidskriftsartikel (refereegranskat)abstract
- Background: The inhibitors that belong to the serpin family are widely distributed regulatory molecules that include most protease inhibitors found in blood. It is generally thought that serpin inhibition involves reactive-centre cleavage, loop insertion and protease translocation, but different models of the serpin–protease complex have been proposed. In the absence of a spatial structure of a serpin–protease complex, a detailed understanding of serpin inhibition and the character of the virtually irreversible complex have remained controversial.Results: We used a recently developed method for making precise distance measurements, based on donor–donor energy migration (DDEM), to accurately triangulate the position of the protease urokinase-type plasminogen activator (uPA) in complex with the serpin plasminogen activator inhibitor type 1 (PAI-1). The distances from residue 344 (P3) in the reactive-centre loop of PAI-1 to residues 185, 266, 313 and 347 (P1′) were determined. Modelling of the complex using this distance information unequivocally placed residue 344 in a position at the distal end from the initial docking site with the reactive-centre loop fully inserted into β sheet A. To validate the model, seven single cysteine substitution mutants of PAI-1 were used to map sites of protease–inhibitor interaction by fluorescence depolarisation measurements of fluorophores attached to these residues and cross-linking using a sulphydryl-specific cross-linker.Conclusions: The data clearly demonstrate that serpin inhibition involves reactive-centre cleavage followed by full-loop insertion whereby the covalently linked protease is translocated from one pole of the inhibitor to the opposite one.
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