| 1. |
- Marushchak, Denys, et al.
(författare)
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Fluorescence Depolarisation Studies of Filamentous Actin Analysed with a Genetic Algorithm
- 2007
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Ingår i: Biophysical Journal. - : CellPress. - 0006-3495 .- 1542-0086. ; 93:9, s. 3291-3299
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Tidskriftsartikel (refereegranskat)abstract
- A new method, in which a genetic algorithm was combined with Brownian dynamics and Monte Carlo simulations, was developed to analyze fluorescence depolarization data collected by the time-correlated single photon-counting technique. It was applied to studies of BODIPY-labeled filamentous actin (F-actin). The technique registered the local order and reorienting motions of the fluorophores, which were covalently coupled to cysteine 374 (C374) in actin and interacted by electronic energy migration within the actin polymers. Analyses of F-actin samples composed of different fractions of labeled actin molecules revealed the known helical organization of F-actin, demonstrating the usefulness of this technique for structure determination of complex protein polymers. The distance from the filament axis to the fluorophore was found to be considerably less than expected from the proposed position of C374 at a high filament radius. In addition, polymerization experiments with BODIPY-actin suggest a 25-fold more efficient signal for filament formation than pyrene-actin.
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| 2. |
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| 3. |
- Bergström, Fredrik, et al.
(författare)
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Dimers of Dipyrrometheneboron Difluoride (BODIPY) with Light Spectroscopic Applications in Chemistry and Biology
- 2002
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 124:2, s. 196-204
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Tidskriftsartikel (refereegranskat)abstract
- A ground-state dimer (denoted DI) exhibiting a strong absorption maximum at 477 nm ( = 97 000 M-1cm-1) can form between adjacent BODIPY groups attached to mutant forms of the protein, plasminogen activator inhibitor type 1 (PAI-1). No fluorescence from excited DI was detected. A locally high concentration of BODIPY groups was also achieved by doping lipid phases (micelles, vesicles) with BODIPY-labeled lipids. In addition to an absorption band located at about 480 nm, a new weak absorption band is also observed at ca. 570 nm. Both bands are ascribed to the formation of BODIPY dimers of different conformation (DI and DII). Contrary to DI in PAI-1, the DII aggregates absorbing at 570 nm are emitting light observed as a broad band centered at about 630 nm. The integrated absorption band of DI is about twice that of the monomer, which is compatible with exciton coupling within a dimer. The Förster radius of electronic energy transfer between a BODIPY excited monomer and the ground-state dimer (DI) is 57 ± 2 Å. A simple model of exciton coupling suggests that in DI two BODIPY groups are stacked on top of each other in a sandwich-like configuration with parallel electronic transition dipoles. For DII the model suggests that the S0 S1 transition dipoles are collinear. An explanation for the previously reported (J. Am. Chem. Soc. 1994, 116, 7801) exceptional light spectroscopic properties of BODIPY is also presented. These are ascribed to the extraordinary electric properties of the BODIPY chromophore. First, changes of the permanent electric dipole moment ( -0.05 D) and polarizability (-26 × 10-40 C m2 V-1) between the ground and the first excited states are small. Second, the S0 S1 electronic transition dipole moments are perpendicular to .
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| 4. |
- Burghart, Armin, et al.
(författare)
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Energy transfer cassettes based on BODIPY® dyes
- 2000
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Ingår i: Chemical Communications. - : Royal Society of Chemistry (RSC). - 1359-7345 .- 1364-548X. ; :2203-4
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Tidskriftsartikel (refereegranskat)abstract
- The donor–acceptor dye cassettes 1–4 were designed to capture energy at a single wavelength and to convert it to well-resolved, intense fluorescence emissions; in practice, Stokes shifts of 40–148 nm, quantum yields of 0.12–0.60, and efficient energy transfer was demonstrated.
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| 5. |
- Chorell, Erik, et al.
(författare)
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Design and Synthesis of Fluorescent Pilicides and Curlicides : Bioactive Tools to Study Bacterial Virulence Mechanisms
- 2012
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Ingår i: Chemistry - A European Journal. - Berlin : Wiley-VCH Verlagsgesellschaft. - 0947-6539 .- 1521-3765. ; 18:15, s. 4522-4532
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Tidskriftsartikel (refereegranskat)abstract
- Pilicides and curlicides are compounds that block the formation of the virulence factors pili and curli, respectively. To facilitate studies of the interaction between these compounds and the pili and curli assembly systems, fluorescent pilicides and curlicides have been synthesized. This was achieved by using a strategy based on structure-activity knowledge, in which key pilicide and curlicide substituents on the ring-fused dihydrothiazolo 2-pyridone central fragment were replaced by fluorophores. Several of the resulting fluorescent compounds had improved activities as measured in pili- and curli-dependent biofilm assays. We created fluorescent pilicides and curlicides by introducing coumarin and 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) fluorophores at two positions on the peptidomimetic pilicide and curlicide central fragment. Fluorescence images of the uropathogenic Escherichia coli (UPEC) strain UTI89 grown in the presence of these compounds shows that the compounds are strongly associated with the bacteria with a heterogeneous distribution.
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| 6. |
- Chorell, Erik, 1980-, et al.
(författare)
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Design and synthesis of fluorescently labeled pilicides and curlicides: bioactive tools to study bacterial virulence mechanisms
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Pilicides and curlicides block formation of the E. coli virulence factors pili and curli. To facilitate studies of the interaction between these compounds and the pili and curli assembly systems, fluorescent pilicides and curlicides have been synthesized. This was achieved using a strategy where key pilicide and curlicide substituents were replaced by fluorophores having similar physicochemical properties. The resulting fluorescent compounds had improved anti-virulence activities as measured in pili- and curli-dependent biofilm assays. We created fluorescent pilicides and curlicides by introducing both coumarin and 4,4-Difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) fluorophores at two positions on the peptidomimetic pilicide and curlicide scaffold. Fluorescence images of the uropathogenic Escherichia coli (UPEC) strain UTI89 grown in the presence of these compounds shows that the compounds are strongly associated to the bacteria and seem to discriminate between different bacteria in a population.
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| 7. |
- 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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| 8. |
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| 9. |
- Edman, Peter, et al.
(författare)
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On determining intramolecular distances from donor–donor energy migration (DDEM) within bifluorophoric macromolecules
- 2000
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Ingår i: PHYSICAL CHEMISTRY CHEMICAL PHYSICS. - : Royal Society of Chemistry (RSC). - 1463-9076 .- 1463-9084. ; 2, s. 1789-94
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Tidskriftsartikel (refereegranskat)abstract
- Recently a model based on donor–donor energy migration (DDEM) was developed for examining structure–function properties of biomacromolecules such as proteins (J. Chem. Soc., Faraday Trans., 1996, 92, 1563). Unlike the extended Förster theory (EFT; J. Chem. Phys., 1996, 105, 10896) the DDEM model is straightforward to apply in the analyses of fluorescence depolarisation experiments, as obtained by the time-correlated single photon counting (TCSPC) technique. In order to test the validity of the DDEM model, the EFT was used to create synthetic depolarisation data. These mimic true TCSPC experiments and cover a wide range of physical conditions, which are difficult to arrange for in real experiments with model systems. In particular, the relative rate of DDEM () and the rotation correlation time () of the donor molecules was examined. The DDEM rates obtained from the analyses were compared to the true rates. From these results the relative error of the intramolecular distances were calculated. For values 1<12<25, the DDEM model is slightly overestimating the distances. Typically, the distances determined with the DDEM model are overestimated by 5–10%.
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| 10. |
- Fa, M, et al.
(författare)
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Conformational studies of plasminogen activator inhibitor type 1 by fluorescence spectroscopy. Analysis of the reactive centre of inhibitory and substrate forms, and of their respective reactive-centre cleaved forms.
- 2000
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Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 267:12, s. 3729-34
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Tidskriftsartikel (refereegranskat)abstract
- The inhibitors that belong to the serpin family are suicide inhibitors that control the major proteolytic cascades in eucaryotes. Recent data suggest that serpin inhibition involves reactive centre cleavage followed by loop insertion, whereby the covalently linked protease is translocated away from the initial docking site. However under certain circumstances, serpins can also be cleaved like a substrate by target proteases. In this report we have studied the conformation of the reactive centre of plasminogen activator inhibitor type 1 (PAI-1) mutants with inhibitory and substrate properties. The polarized steady-state and time-resolved fluorescence anisotropies were determined for BODIPY(R) probes attached to the P1' and P3 positions of the substrate and active forms of PAI-1. The fluorescence data suggest an extended orientational freedom of the probe in the reactive centre of the substrate form as compared to the active form, revealing that the conformation of the reactive centres differ. The intramolecular distance between the P1' and P3 residues in reactive centre cleaved inhibitory and substrate mutants of PAI-1, were determined by using the donor-donor energy migration (DDEM) method. The distances found were 57+/-4 A and 63+/-3 A, respectively, which is comparable to the distance obtained between the same residues when PAI-1 is in complex with urokinase-type plasminogen activator (uPA). Following reactive centre cleavage, our data suggest that the core of the inhibitory and substrate forms possesses an inherited ability of fully inserting the reactive centre loop into beta-sheet A. In the inhibitory forms of PAI-1 forming serpin-protease complexes, this ability leads to a translocation of the cognate protease from one pole of the inhibitor to the opposite one.
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