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  • 1.
    Bergström, Fredrik
    et al.
    Umeå University, Faculty of Science and Technology, Chemistry.
    Mikhalyov, Ilya
    Umeå University, Faculty of Science and Technology, Chemistry.
    Hägglöf, Peter
    Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Wortmann, Rüdiger
    Ny, Tor
    Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Johansson, Lennart B-Å
    Dimers of Dipyrrometheneboron Difluoride (BODIPY) with Light Spectroscopic Applications in Chemistry and Biology2002In: Journal of the American Chemical Society, Vol. 124, no 2, p. 196-204Article in journal (Refereed)
    Abstract [en]

    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 .

  • 2. Burghart, Armin
    et al.
    Thoresen, Lars H
    Chen, Jiong
    Burgess, Kevin
    Bergström, Fredrik
    Umeå University, Faculty of Science and Technology, Chemistry.
    Johansson, Lennart B-Å
    Umeå University, Faculty of Science and Technology, Chemistry.
    Energy transfer cassettes based on BODIPY® dyes2000In: Chemical Communications, no 2203-4Article in journal (Refereed)
    Abstract [en]

    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.

  • 3.
    Chorell, Erik
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Pinkner, Jerome S
    Bengtsson, Christoffer
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Edvinsson, Sofie
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Cusumano, Corinne K
    Rosenbaum, Erik
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Johansson, Lennart B-Å
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Hultgren, Scott J
    Almqvist, Fredrik
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Design and Synthesis of Fluorescent Pilicides and Curlicides: Bioactive Tools to Study Bacterial Virulence Mechanisms2012In: Chemistry - A European Journal, ISSN 0947-6539, E-ISSN 1521-3765, Vol. 18, no 15, p. 4522-4532Article in journal (Refereed)
    Abstract [en]

    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.

  • 4.
    Chorell, Erik
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Pinkner, Jerome S.
    Department of Molecular Microbiology, Washington University, School of Medicine, St. Louis, Missouri 63110, USA.
    Bengtsson, Christoffer
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Edvinsson, Sofie
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Cusumano, Corinne K.
    Rosenbaum, Erik
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Johansson, Lennart B-Å
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Hultgren, Scott J.
    Department of Molecular Microbiology, Washington University, School of Medicine, St. Louis, Missouri 63110, USA.
    Almqvist, Fredrik
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Design and synthesis of fluorescently labeled pilicides and curlicides: bioactive tools to study bacterial virulence mechanismsManuscript (preprint) (Other academic)
    Abstract [en]

    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.

  • 5.
    Edman, Peter
    et al.
    Umeå University, Faculty of Science and Technology, Chemistry.
    Bergström, Fredrik
    Umeå University, Faculty of Science and Technology, Chemistry.
    Johansson, Lennart B-Å
    Umeå University, Faculty of Science and Technology, Chemistry.
    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 systems2000In: Physical chemistry chemical physics, Vol. 2, no 12, p. 2795-2801Article in journal (Refereed)
    Abstract [en]

    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.

  • 6.
    Edman, Peter
    et al.
    Umeå University, Faculty of Science and Technology, Chemistry.
    Westlund, Per-Olof
    Umeå University, Faculty of Science and Technology, Chemistry.
    Johansson, Lennart B-Å
    Umeå University, Faculty of Science and Technology, Chemistry.
    On determining intramolecular distances from donor–donor energy migration (DDEM) within bifluorophoric macromolecules2000In: PHYSICAL CHEMISTRY CHEMICAL PHYSICS, Vol. 2, p. 1789-94Article in journal (Refereed)
    Abstract [en]

    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%.

  • 7.
    Fa, Ming
    et al.
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Bergström, Fredrik
    Faculty of Science and Technology, Chemistry.
    Hägglöf, Peter
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Wilczynska, Malgorzata
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Johansson, Lennart B-Å
    Faculty of Science and Technology, Chemistry.
    Ny, Tor
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    The structure of a serpin–protease complex revealed by intramolecular distance measurements using donor–donor energy migration and mapping of interaction sites2000In: Structure, Vol. 8, no 4, p. 397-405Article in journal (Refereed)
    Abstract [en]

    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.

  • 8.
    Fa, Ming
    et al.
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Bergström, Fredrik
    Faculty of Science and Technology, Chemistry.
    Johansson, Lennart B-Å
    Faculty of Science and Technology, Chemistry.
    Ny, Tor
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    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 forms2000In: European Journal of Biochemistry, Vol. 267, no 12, p. 3729-34Article in journal (Refereed)
    Abstract [en]

    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® 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 Å and 63 ± 3 Å, 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 β-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.

  • 9. Habenicht, Anja
    et al.
    Hjelm, Johan
    Mukhtar, Emad
    Bergström, Fredrik
    Umeå University, Faculty of Science and Technology, Chemistry.
    Johansson, Lennart B-Å
    Umeå University, Faculty of Science and Technology, Chemistry.
    Two-photon excitation and time-resolved fluorescence: I. The proper response function for analysing single-photon counting experiments2002In: Chemical Physics Letters, Vol. 354, no 5-6, p. 367-75Article in journal (Refereed)
    Abstract [en]

    An accurate instrumental response function is needed to deconvolute fluorescence data obtained by time-correlated single-photon counting (TCSPC) upon multi-photon excitation. Hitherto the response function was obtained by measuring Rayleigh scattering (RS) from colloidal solutions, as is also used in one-photon excited fluorescence. We show that hyper Rayleigh scattering (HRS) provides a better choice for deconvolution of fluorescence decays, as obtained by TCSPC and two-photon excitation (TPE). The one- and two-photon response functions were monitored as RS and HRS from colloidal gold particles at 800 and 400 nm, respectively.

  • 10.
    Humpolickova, Jana
    et al.
    J. Heyrovský Institute of Physical Chemistry, Prague, Czech Republic.
    Stefl, Martin
    J. Heyrovský Institute of Physical Chemistry, Prague, Czech Republic.
    Sachl, Radek
    J. Heyrovský Institute of Physical Chemistry, Prague, Czech Republic.
    Cebecauer, Marek
    J. Heyrovský Institute of Physical Chemistry, Prague, Czech Republic.
    Machan, Radek
    J. Heyrovský Institute of Physical Chemistry, Prague, Czech Republic.
    Johansson, Lennart B-Å
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Hof, Martin
    J. Heyrovský Institute of Physical Chemistry, Prague, Czech Republic.
    Dynamics and size of crosslinking-induced lipid nanodomains in model membranes2012In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 102, no 3, p. 294a-Article in journal (Refereed)
    Abstract [en]

    Changes of membrane organization upon crosslinking of its components trigger cellsignaling response to various exogenous factors. Crosslinking of raft gangliosides GM1with cholera toxin (CTxB) was demonstrated to cause microscopic phase separation inmodel membranes and the CTxB-GM1 complexes forming a minimal lipid raft unit aresubject of ongoing cell membrane research. Yet, those subdiffraction sized rafts havenever been described in terms of size and dynamics. By means of two-color z-scanfluorescence correlation spectroscopy, we show that the nano-sized domains are formedin model membranes at lower sphingomyelin content than needed for the large scalephase separation and that the CTxB-GM1 complexes are confined in the domains poorlystabilized with sphingomyelin. Fluorescence resonance energy transfer together withMonte Carlo modeling of the donor decay response reveal the domain radius ofapproximately 8 nm, which increases at higher sphingomyelin content. We observed twotypes of differently behaving domains, which suggests a dual role of the crosslinker: first,local transient condensation of the GM1 molecules compensating lack of sphingomyelinand second, coalescence of existing nanodomains ending in large scale phase separation.

  • 11.
    Hägglöf, Peter
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Bergström, Fredrik
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wilczynska, Malgorzata
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Johansson, Lennart B-Å
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    The reactive-center loop of active PAI-1 is folded close to the protein core and can be partially inserted2004In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 335, no 3, p. 823-832Article in journal (Refereed)
    Abstract [en]

    Plasminogen activator inhibitor 1 (PAI-1) is the main inhibitor of plasminogen activators and plays an important role in many pathophysiological processes. Like other members of the serpin family, PAI-1 has a reactive center consisting of a mobile loop (RCL) with P1 and P1′ residues acting as a “bait” for cognate protease. In contrast to the other serpins, PAI-1 loses activity by spontaneous conversion to an inactive latent form. This involves full insertion of the RCL into β-sheet A. To search for molecular determinants that could be responsible for conversion of PAI-1 to the latent form, we studied the conformation of the RCL in active PAI-1 in solution. Intramolecular distance measurements by donor–donor energy migration and probe quenching methods reveal that the RCL is located much closer to the core of PAI-1 than has been suggested by the recently resolved X-ray structures of stable PAI-1 mutants. Disulfide bonds can be formed in double-cysteine mutants with substitutions at positions P11 or P13 of the RCL and neighboring residues in β-sheet A. This suggests that the RCL may be preinserted up to residue P13 in active PAI-1, and possibly even to residue P11. We propose that the close proximity of the RCL to the protein core, and the ability of the loop to preinsert into β-sheet A is a possible reason for PAI-1 being able to convert spontaneously to its latent form.

  • 12.
    Håkansson, Pär
    et al.
    Umeå University, Faculty of Science and Technology, Chemistry.
    Isaksson, Mikael
    Umeå University, Faculty of Science and Technology, Chemistry. Umeå University, Faculty of Science and Technology, Chemistry.
    Westlund, Per-Olof
    Umeå University, Faculty of Science and Technology, Chemistry.
    Johansson, Lennart B-Å
    Umeå University, Faculty of Science and Technology, Chemistry.
    Extended Förster theory for determining intraprotein distances: 1. The K2-dynamics and fluorophore reorientation2004In: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 108, no 44, p. 17243-17250Article in journal (Refereed)
    Abstract [en]

    A detailed analysis of the previously developed (J. Chem. Phys. 1996, 105, 10896) extended Förster theory (EFT) is presented for analyzing electronic energy migration within pairs of donors (D). Synthetic data that mimics experimental time-correlated single photon counting data were generated and re-analyzed. To cover a wide dynamic range and various orientational restrictions, the rates of reorientation, as well as the orientational configurations of the interacting D-groups were varied. In general DD distances are recovered within an error limit of 5%, while the errors in orientational configurations are usually larger. The Maier−Saupe and cone potentials were used to generate an immense variety of orientational trajectories. The results obtained exhibit no significant dependence on the choice of potential function used for generating EFT data. Present work demonstrates how to overcome the classical “κ2-problem” and the frequently applied approximation of κ2 = 2/3 in the data analyses. This study also outlines the procedure for analyzing fluorescence depolarization data obtained for proteins, which are specifically labeled with D-groups. The EFT presented here brings the analyses of DDEM data to the same level of molecular detail as in ESR- and NMR-spectroscopy.

  • 13.
    Isaksson, Mikael
    et al.
    Umeå University, Faculty of Science and Technology, Chemistry.
    Hägglöf, Peter
    Håkansson, Pär
    Umeå University, Faculty of Science and Technology, Chemistry.
    Ny, Tor
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Johansson, Lennart B-Å
    Umeå University, Faculty of Science and Technology, Chemistry.
    Extended Förster theory for determining intraprotein distances: 2. an accurate analysis of fluorescence depolarisation experiments2007In: Physical Chemistry, Chemical Physics - PCCP, ISSN 1463-9076, E-ISSN 1463-9084, Vol. 9, p. 3914-3922Article in journal (Refereed)
    Abstract [en]

    The extended Förster theory (EFT) is for the first time applied to the quantitative determination of the intramolecular distances in proteins. It is shown how the EFT (J. Chem. Phys., 1996, 105, 10896) can be adapted to the analyses of fluorescence depolarisation experiments based on the time-correlated single photon counting technique (TCSPC). The protein system studied was the latent form of plasminogen activator inhibitor type I (PAI-1), which was mutated and labelled by the thiol reactive BODIPY® derivative {N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-yl)methyl iodoacetamide}. The energy migration occurs within pairs of photophysically identical donor groups that undergo reorientational motions on the timescales of energy migration and fluorescence relaxation. Unlike all models currently used for analysing fluorescence TCSPC data, the EFT explicitly accounts for the time-dependent reorientations that influence the rate of electronic energy transfer/migration in a complex manner. The complexity is related to the 2 problem, which has been discussed for years. The EFT brings the analyses of DDEM data to the same level of molecular description as in ESR and NMR spectroscopy, i.e. it yields microscopic information about the reorientation correlation times, the order parameters, as well as inter-chromophoric distances.

  • 14.
    Isaksson, Mikael
    et al.
    Umeå University, Faculty of Science and Technology, Chemistry.
    Kalinin, Stanislav
    Umeå University, Faculty of Science and Technology, Chemistry.
    Lobov, Sergei
    Umeå University, Faculty of Science and Technology, Chemistry.
    Ny, Tor
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Johansson, Lennart B-Å
    Umeå University, Faculty of Science and Technology, Chemistry.
    An environmental-sensitive BODIPY®-derivative with bioapplication: spectral and photophysical properties2003In: Journal of Fluorescence, ISSN 1053-0509 (Print) 1573-4994 (Online), Vol. 13, no 5, p. 379-84Article in journal (Refereed)
    Abstract [en]

    A previously synthesised derivative of BODIPY aimed for sulfhydryl specific labelling of cysteine residues in proteins was studied. The spectral and photophysical properties of this derivative, N-(4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene-2-yl) iodoacetamide (NBDY) were characterised, and found to be considerably different from those of commonly used derivatives of BODIPY, e.g. N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-yl)methyl iodoacetamide. The absorption and fluorescence spectra, as well as fluorescence lifetimes and quantum yields of NBDY are quite sensitive to solvent properties. The fluorescence is effectively quenched by I– when NBDY is free in water or attached to Cys in different mutants of plasminogen activator inhibitor type 2 (PAI-2). A ground-state dimer forms when two NBDY groups are closely spaced in plasminogen activator inhibitor type 1 (PAI-1).

  • 15.
    Isaksson, Mikael
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Kalinin, Stanislav
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Lobov, Sergei
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Wang, Shouye
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Johansson, Lennart B-Å
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Partial donor-donor energy migration (PDDEM): a novel fluorescence method for internal protein distance measurements2004In: Physical Chemistry, Chemical Physics - PCCP, ISSN 1463-9076, E-ISSN 1463-9084, Vol. 6, no 11, p. 3001-3008Article in journal (Refereed)
    Abstract [en]

    We show that the photophysics of chemically identical but photophysically non-identical fluorescent pairs can be used for measuring distances within proteins. For this purpose, the theory of partial donor-donor energy migration (PDDEM, S. Kalinin, J. G. Molotkovsky and L. B.-Angstrom. Johansson, Spectrochim. Acta, Part A, 2002, 58, 1057-1097) was applied for distance measurements between BODIPY groups covalently linked to cystein residues in plasminogen activator inhibitor of type 2 (PAI-2). Two sulfhydryl specific derivatives of BODIPY were used namely: N-(4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene-2-yl) iodoacetamide and N-(4.4-difluoro-5.7-ditriethyl-4-bona-3a,4a-diaza-s-indacene-3-yl) methyl iodoacetamide. To determine distances, the time-resolved fluorescence relaxation for two singly labelled forms of PAI-2, as well as the corresponding doubly labelled protein were combined and analysed in a global manner. Fluorescence depolarisation experiments on the labelled mutants were also analysed. The distances determined by PDDEM were in good agreement to those obtained from donor-donor energy migration (DDEM) experiments and structural data on PAI-2. The PDDEM approach allows for the use of very different fluorescent probes, which enables wide range of distances to be measured. The PDDEM model also provides a rational explanation to why previous observations of polyfluorophore-labelled proteins exhibit a shorter average fluorescence lifetime compared to the arithmetic average of lifetimes obtained for the corresponding single labelled proteins.

  • 16.
    Isaksson, Mikael
    et al.
    Umeå University, Faculty of Science and Technology, Chemistry.
    Norlin, Nils
    Umeå University, Faculty of Science and Technology, Chemistry.
    Westlund, Per-Olof
    Umeå University, Faculty of Science and Technology, Chemistry.
    Johansson, Lennart B-Å
    Umeå University, Faculty of Science and Technology, Chemistry.
    On the quantitative molecular analysis of electronic energy transfer within donor–acceptor pairs2007In: Physical Chemistry, Chemical Physics - PCCP, ISSN 1463-9076, E-ISSN 1463-9084, Vol. 9, p. 1941-51Article in journal (Refereed)
    Abstract [en]

    An extended Förster theory (EFT) on electronic energy transfer is presented for the quantitative analysis of time-resolved fluorescence lifetime and depolarisation experiments. The EFT, which was derived from the stochastic Liouville equation, yields microscopic information concerning the reorientation correlation times, the order parameters, as well as inter chromophoric distances. Weakly interacting donor and acceptor groups, which reorient and interact in a pair wise fashion, are considered, under isotropic and anisotropic conditions. For the analysis of experiments it is shown that not only do we need to consider the orientational distributions of the transition dipoles, but the internal reorienting molecular dynamics within the pair which is of even greater importance. The latter determines the shape as well as the rate of the observed donor fluorescence and depolarisation decays, which are most often not mono-exponential functions. It is shown that the commonly used Förster theory is a special case of the EFT. Strategies are presented for applying the EFT, which makes use of Brownian dynamics simulation.

  • 17. Jiao, Guan-Sheng
    et al.
    Loudet, Aurore
    Lee, Hong Boon
    Kalinin, Stanislav
    Umeå University, Faculty of Science and Technology, Chemistry.
    Johansson, Lennart B-Å
    Umeå University, Faculty of Science and Technology, Chemistry.
    Burgess, Kevin
    Syntheses and spectroscopic properties of energy transfer systems based on squaraines2003In: Tetrahedron, Vol. 59, no 17, p. 3109-16Article in journal (Refereed)
    Abstract [en]

    The purpose of this project was to prepare fluorescent dyes that could absorb energy at relatively short wavelengths, and fluoresce in the near-IR region. To achieve this, copper- and palladium-mediated C–N couplings were used to prepare the ‘cassettes’, i.e the carbazole derivative 3b and the carbazole-, phenothiazine-, and phenoazine-squaraines 4b–d. These compounds have carbazole, phenothiazine, and phenoazine donor-components that absorb around about 300–320 nm, and squaraine acceptor-parts that fluoresce in the range 650–700 nm. The efficiencies of energy transfer from the donor to the acceptor, and the overall quantum yields of the cassettes were determined.

  • 18.
    Johansson, Lennart B-Å
    Umeå University, Faculty of Science and Technology, Chemistry.
    Modern fluorescence spectroscopy: Insights into biosystems at a molecular level - Preface2001In: Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, Vol. 57, no 11, p. 2091-Article in journal (Refereed)
  • 19.
    Kalinin, Stanislav
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Johansson, Lennart B-Å
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Energy migration and transfer rates are invariant to modeling the fluorescence relaxation by discrete and continuous distributions of lifetimes2004In: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, ISSN 1520-6106, Vol. 108, no 9, p. 3092-3097Article in journal (Refereed)
    Abstract [en]

    Fluorescent groups typically exhibit nonexponential photophysics when incorporated into biomolecular structures, e.g., proteins and lipid membranes. Models assuming discrete and continuous distributions of lifetimes can each accurately describe the observed relaxation. In the analyses of energy transfer and PDDEM (partial donor-donor energy migration) experiments, one frequently needs to model the nonexponential decays of noninteracting donor and acceptor groups. The present paper aims at exploring whether calculated transfer/migration rates depend on modeling the photophysics' decay by discrete or continuous distributions of lifetimes. Discrete or continuous distribution models of the decay, generated synthetically, were analyzed as well as true experimental data. Two proteins were studied. In one of the systems, we examined energy transfer from Trp (donor) to BODIPY (acceptor) in ribosomal protein S6, obtained from Thermus thermophilus. In the second system, we examined PDDEM between different BODIPY derivatives that were pairwise specifically incorporated in mutant forms of plasminogen activator inhibitor type 2. Interestingly, the rates of electronic energy migration/transfer and distances determined within pairs of interacting chromophores reveal small or negligible influence on using discrete or continuous distributions of lifetimes.

  • 20.
    Kalinin, Stanislav
    et al.
    Umeå University, Faculty of Science and Technology, Chemistry.
    Johansson, Lennart B-Å
    Umeå University, Faculty of Science and Technology, Chemistry.
    Utility and Considerations of Donor–Donor Energy Migration as a Fluorescence Method for Exploring Protein Structure-Function2004In: Journal of Fluorescence, ISSN 1053-0509 (Print) 1573-4994 (Online), Vol. 14, no 6, p. 681-91Article in journal (Refereed)
    Abstract [en]

    This review aims at surveying the use of electronic energy transport between chemically identical fluorophores (i.e. donors) in studies of various protein systems. Applications of intra- and interprotein energy migration are presented that make use of polarised steady-state and time-resolved fluorescence spectroscopic techniques. The donor-donor energy migration (DDEM) and the partial donor-donor energy migration (PDDEM) models for calculating distances between donor groups are exposed together with the most recent development of an extended Förster theory (EFT). Synthetic fluorescence depolarisation data that mimic time-correlated single photon counting experiments were generated using the EFT, and then further re-analysed by the different models. The results obtained were compared with the known parameters used to generate EFT data. Aspects on how to adopt the EFT in the analyses of time-correlated single photon counting experiments are also presented, as well as future aspects on using energy migration for examining protein structure.

  • 21.
    Kalinin, Stanislav
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Molotkovsky, Julian G
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Johansson, Lennart B-Å
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Distance Measurements Using Partial Donor-Donor Energy Migration within Pairs of Fluorescent Groups in Lipid Bilayers.2003In: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 107, no 14, p. 3318-3324Article in journal (Refereed)
    Abstract [en]

    For distance measurements the fluorescence relaxation was explored within pairwise interacting chemically identical, but photophysically different fluorescent groups. The recently developed theory (Kalinin, S. V.; et al. Spectrochim. Acta, Part A 2002, 58, 1087-1097) of partial donor-donor energy migration (PDDEM) was tested on lipid vesicles as a model system, which, for example, enables arrangement of a pH gradient between the inside and outside of the lipid bilayer. Time-correlated single-photon counting and steady-state fluorescence experiments were performed on mono- and bis-lipid derivatives of fluorescein solubilized in lipid bilayers. The PDDEM approach was successfully applied in the analyses of the different experiments. The distances extracted between the fluorescein groups of bis-fluorescein derivatives in lipid vesicles composed 1,2-dioleoyl-sn-glycero-3-phosphocholine and 1,2-dimyristoyl-sn-glycero-3-phosphocholine were found to be in reasonable agreement with independent measurements of the bilayer thickness.

  • 22. Kalinin, Stanislav
    et al.
    Speckbacher, Marcus
    Langhals, Heinz
    Johansson, Lennart B-Å
    Umeå University, Faculty of Science and Technology, Chemistry.
    A new and versatile fluorescence standard for quantum yield determination2001In: PHYSICAL CHEMISTRY CHEMICAL PHYSICS, ISSN 1463-9076, Vol. 3, no 2, p. 172-4Article in journal (Refereed)
    Abstract [en]

    It is demonstrated that a new bichromophoric compound, N-2,N-3-[bis(1-hexylheptyl)-benzo[ghi]perylene-2,3,8,9,11,12-hexacarboxylic-2,3:8,9:11,12-tris(dicarboximide)]-N-1,N-1'-(1,2-ethyl)-[N-2'-(1-octylnonyl)-perylene-3,4:9,10-bis(dicarboximide)] (denoted by 1-2), provides a versatile reference molecule for measurements of fluorescence quantum yields. The compound consists of a donor (1) and an acceptor (2) part for the electronic energy. Absorption spectra of 1-2 form a nearly perfect superposition of the corresponding spectra for 1 and 2. In addition to a fluorescence quantum yield of one, 1-2 allows for choosing excitation wavelengths between 300 and 530 nm. The fluorescence lifetime of 1-2 is 4.0 +/- 0.1 ns and is independent of the excitation wavelength. The fluorescence excitation anisotropy reveals that the S-0 --> S-1 and S-0 --> S-2 transition dipoles of 1 are preferentially polarised parallel and perpendicular, respectively, to the C-2-symmetry axis of 1. The rapid transfer of electronic energy is compatible with energy transfer of the Forster type.

  • 23. Kessel, Line
    et al.
    Kalinin, Stanislav
    Umeå University, Faculty of Science and Technology, Chemistry.
    Soroka, Vladislav
    Larsen, Michael
    Johansson, Lennart B-Å
    Umeå University, Faculty of Science and Technology, Chemistry.
    Impact of UVR-A on whole human lenses, supernatants of buffered human lens homogenates, and purified argpyrimidine and 3-OH-kynurenine2005In: ACTA OPHTHALMOLOGICA SCANDINAVICA, ISSN 1395-3907, Vol. 83, no 2, p. 221-7Article in journal (Refereed)
    Abstract [en]

    Purpose: Yellow chromophores and fluorescent compounds accumulate in the lens with age. Some of these compounds are photochemically active. The present study aimed to examine the photochemical effect of ultraviolet radiation-A (UVR-A) on the human lens.

    Methods: Intact human lenses and supernatants of buffered lens homogenates were exposed to UVR-A. The effect of UVR-A was evaluated by time-resolved and steady-state fluorescence spectroscopy, visual evaluation of colour and protein gel electrophoresis.

    Results: Intact lenses exposed to UVR-A showed no changes in time-resolved or steady-state fluorescence properties but the yellow coloration was visibly attenuated. The supernatants of buffered lens homogenates exposed to UVR-A demonstrated a reduction in time-resolved and steady-state fluorescent properties and protein cross-linking.

    Conclusions: Exposure of the intact lens to UVR-A causes chromophore bleaching without affecting fluorescence, indicating that non-fluorescent chromophores have been destroyed. After homogenization, both chromophores and fluorophores from the lens suffer damage and proteins aggregate. This indicates that powerful mechanisms of protection against UVR-A found in the intact lens are disturbed by homogenization of the lens, suggesting that isolated lens proteins cannot be used as a model system for studying cataractogenesis. Hypothetically, the protective mechanism could be related to the rigidly packed three-dimensional structure of the lens proteins or to the abundance of antioxidative and free radical scavenging defence systems.

  • 24.
    Krishnan, K. Syam
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Bengtsson, Christoffer
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Good, James A. D.
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Mirkhanov, Shamil
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Chorell, Erik
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Johansson, Lennart B. -Å.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Almqvist, Fredrik
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Synthesis of fluorescent ring-fused 2-pyridone peptidomimetics2013In: Journal of Organic Chemistry, ISSN 0022-3263, E-ISSN 1520-6904, Vol. 78, no 23, p. 12207-12213Article in journal (Refereed)
    Abstract [en]

    Thiazolino fused 2-pyridones peptidomimetics are of significant biological importance due to their ability to interfere with adhesive fiber formation in uropathogenic Escherichia coli and oligomerization of amyloid fibres. We have developed an efficient synthetic route to fluorescent BODIPY analogues, with structural diversification from a key intermediate enabling introduction of C-2 substituents and late incorporation of the BODIPY moiety. A mild lithium halide mediated hydrolysis enabled preparation of peptidomimetic fluorophores with useful photophysical properties for further chemical biology applications.

  • 25. Langhals, Heinz
    et al.
    Walter, Andreas
    Rosenbaum, Erik
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Johansson, Lennart B-Å
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    A versatile standard for bathochromic fluorescence based on intramolecular FRET2011In: Physical Chemistry, Chemical Physics - PCCP, ISSN 1463-9076, E-ISSN 1463-9084, Vol. 13, no 23, p. 11055-11059Article in journal (Refereed)
    Abstract [en]

    A perylene and a terrylene tetracarboxylic bisimide dyad was prepared in which an efficient energy transfer from the former to the latter is observed. The absorption spectrum of this compound covers a broad range. Bathochromic fluorescence with a high quantum yield was obtained independent of excitation wavelengths (λ < 655 nm). The dyad can be recommended for the use of calibrating fluorescence spectrometers, as well as a fluorescence standard in the bathochromic region.

  • 26.
    Lobov, Sergei
    et al.
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Wilczynska, Malgorzata
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Bergström, Fredrik
    Faculty of Science and Technology, Chemistry.
    Johansson, Lennart B-Å
    Faculty of Science and Technology, Chemistry.
    Ny, Tor
    Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
    Structural Bases of the Redox-dependent Conformational Switch in the Serpin PAI-22004In: Journal of Molecular Biology, Vol. 344, no 5, p. 1359-68Article in journal (Refereed)
    Abstract [en]

    Depending on the redox-status, the serpin plasminogen activator inhibitor type 2 (PAI-2) can exist in either a stable monomeric or polymerogenic form. The latter form, which spontaneously forms loop-sheet polymers, has an open β-sheet A and is stabilized by a disulfide bond between C79 (in the CD-loop) and C161 (at the bottom of PAI-2). Reduction of this bond results in a closing of the β-sheet A and converts PAI-2 to a stable monomeric form. Here we show that the stable monomeric and polymerogenic forms of PAI-2 are fully interconvertible, depending on redox-status of the environment. Our intramolecular distance measurements indicate that the CD-loop folds mainly on one side of the stable monomeric form of the inhibitor. However, the loop can translocate about 54 Å to the bottom of PAI-2 so that the C79–C161 disulfide bond can form under oxidizing conditions. We show also that the redox-active C79 can form a disulfide-link to the matrix protein vitronectin, suggesting that vitronectin can stabilize active PAI-2 in extracellular compartments. PAI-2 is therefore a rare example of a redox-sensitive protein for which the activity and polymerization ability are regulated by reversible disulfide bond formation leading to major translocation of a loop and significant conformational changes in the molecule.

  • 27.
    Marushchak, Denys
    et al.
    Umeå University, Faculty of Science and Technology, Chemistry.
    Grenklo, Staffan
    Johansson, Thomas
    Karlsson, Roger
    Johansson, Lennart B-Å
    Umeå University, Faculty of Science and Technology, Chemistry.
    Fluorescence Depolarisation Studies of Filamentous Actin Analysed with a Genetic Algorithm2007In: Biophysical Journal, Vol. 93, p. 3291-99Article in journal (Refereed)
    Abstract [en]

    A new method, in which a genetic algorithm (GA) was combined with Brownian dynamics and Monte Carlo simulations, was developed to analyse fluorescence depolarisation data collected by the time-correlated single photon counting technique. It was applied to studies of BODIPY-{N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-yl)methyl}-labelled 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 labelled 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, polymerisation experiments with BODIPY-actin suggest a 25-fold more efficient signal for filament formation than pyrene-actin.

  • 28.
    Marushchak, Denys
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Gretskaya, Natalia
    Shemyakin & Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia.
    Mikhalyov, Ilya
    Shemyakin & Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia.
    Johansson, Lennart B-Å
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Self-aggregation: an intrinsic property of GM1 in lipid bilayers2007In: Molecular membrane biology, ISSN 0968-7688, E-ISSN 1464-5203, Vol. 24, no 2, p. 102-112Article in journal (Refereed)
    Abstract [en]

    We demonstrate that the ganglioside GM1 in lipid bilayers of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) exhibits a non-uniform lateral distribution, i.e., enriched regions of GM1 molecules are formed, which is an argument in favour of self-aggregation of GM1 being an intrinsic property of GM1 ganglioside. This was concluded from energy transfer/migration studies of BODIPY-labelled gangliosides by means of time-resolved fluorescence lifetime and depolarization experiments. Three fluorophore-labelled gangliosides were synthesized to include either of two spectroscopically different BODIPY groups. These were specifically localized either in the polar headgroup region or in the non-polar region of the lipid bilayer. An eventual ganglioside-ganglioside affinity/aggregation induced by the BODIPY groups was experimentally excluded, which suggests their use in examining the influence of GM1 in more complex systems.

  • 29.
    Marushchak, Denys
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Johansson, Lennart B.A.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    On the quantitative treatment of donor-donor energy migration in regularly aggregated proteins2005In: Journal of Fluorescence, ISSN 1053-0509, E-ISSN 1573-4994, Vol. 15, no 5, p. 797-803Article in journal (Refereed)
    Abstract [en]

    An algorithm is presented that quantitatively accounts for donor–donor energy migration (DDEM) among fluorophore-labeled proteins forming regular aggregates. The DDEM algorithm is based on Monte Carlo and Brownian dynamics simulations and applies to calculation of fluorescence depolarisation data, such as the fluorescence anisotropy. Thereby local orientations, as well as reorienting motions of the fluorescent group are considered in the absence and presence of DDEM and among, in principle, infinitely many proteins as they form regular aggregates. Here we apply the algorithm for calculating and illustrating the DDEM and the time-resolved fluorescence anisotropy under static as well as dynamic conditions within helical, linear and circular aggregate structures. A principal approach of the DDEM algorithm for analysing protein aggregates is also outlined.

  • 30.
    Marushchak, Denys
    et al.
    Umeå University, Faculty of Science and Technology, Chemistry.
    Kalinin, Stanislav
    Umeå University, Faculty of Science and Technology, Chemistry.
    Mikhalyov, Ilya
    Umeå University, Faculty of Science and Technology, Chemistry.
    Gretskaya, Natalia
    Johansson, Lennart B-Å
    Umeå University, Faculty of Science and Technology, Chemistry.
    Pyrromethene dyes (BODIPY) can form ground state homo and hetero dimers: photophysics and spectral properties.2006In: Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, ISSN 1386-1425, Vol. 65, no 1, p. 113-22Article in journal (Refereed)
    Abstract [en]

    Homo and hetero dimerisation of two spectroscopically different BODIPY chromophores was studied, namely, 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene and its 5-styryl-derivative. These exhibit very similar absorption and fluorescence spectral shape, but are mutually shifted by ca. 70 nm. For this reason the former and the latter are referred to as the green and red BODIPY, which here are denoted gB and rB, respectively. Various spectroscopic properties of the rB in different common solvents were determined. The calculated and experimental fluorescence quantum yield is found to be close to 100%, the fluorescence relaxation has a single exponential decay with a lifetime of about 4.5 ns, and the Forster radius for donor-donor energy migration is 67+/-1A. The dimerisation in different solvents was examined by using custom synthesised; mono and bis BODIPY-labelled forms of 1,2-cis-diaminocyclohexane. It is shown that gB and rB can form ground state homo- as well as hetero dimers. The dimers are non-fluorescent, compatible with H-dimers and may act as excitation traps or as acceptors to the corresponding excited monomers.

  • 31.
    Mikaelsson, Therese
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Sachl, Radek
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Johansson, Lennart B-Å
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Electronic energy transport and Fluorescence Spectroscopy for structural insights into Proteins, Regular Protein Aggregates and Lipid Systems2009In: Reviews In Fluorescence 2007: Volume 2007, New York: Springer-Verlag , 2009, p. 53-86Chapter in book (Other academic)
    Abstract [en]

    The present review aims at surveying recent theoretical development and applications of electronic energy transport between chromophoric molecules (i.e. donors and acceptors) in various protein and lipid systems. Reversible, partly reversible, and irreversible energy transport within pairs of interacting chromophoric molecules are considered. Also energy migration/transfer within ensembles of many donor and acceptor molecules is discussed. An extended Förster theory of interacting pairs is summarised, which brings the analyses of data to the same level of molecular description as in ESR and NMR spectroscopy. Recent applications of energy transfer/migration on protein systems concern their structure, folding, and their formation of non-covalent protein polymers. The latter systems are of particular interest in e.g. the study of amyloid formation and the molecular functioning of muscles. The energy transfer/migration processes have also been utilised to study the spatial distribution of lipid molecules, which is of interest in the study of biological membranes and their functioning, e.g. the presumed formation of so-called rafts.

  • 32.
    Mikaelsson, Therese
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Ådén, Jörgen
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Johansson, Lennart B-Å
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wittung-Stafshede, Pernilla
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Direct Observation of Protein Unfolded State Compaction in the Presence of Macromolecular Crowding2013In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 104, no 3, p. 694-704Article in journal (Refereed)
    Abstract [en]

    Proteins fold and function in cellular environments that are crowded with other macromolecules. As a consequence of excluded volume effects, compact folded states of proteins should be indirectly stabilized due to destabilization of extended unfolded conformations. Here, we assess the role of excluded volume in terms of protein stability, structural dimensions and folding dynamics using a sugar-based crowding agent, dextran 20, and the small ribosomal protein S16 as a model system. To specifically address dimensions, we labeled the protein with BODIPY at two positions and measured Trp-BODIPY distances under different conditions. As expected, we found that dextran 20 (200 mg/ml) stabilized the variants against urea-induced unfolding. At conditions where the protein is unfolded, Förster resonance energy transfer measurements reveal that in the presence of dextran, the unfolded ensemble is more compact and there is residual structure left as probed by far-ultraviolet circular dichroism. In the presence of a crowding agent, folding rates are faster in the two-state regime, and at low denaturant concentrations, a kinetic intermediate is favored. Our study provides direct evidence for protein unfolded-state compaction in the presence of macromolecular crowding along with its energetic and kinetic consequences.

  • 33.
    Mikaelsson, Therese
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Ådén, Jörgen
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wittung-Stafshede, Pernilla
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Johansson, Lennart B-Å
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Macromolecular crowding effects on two homologs of ribosomal protein S16: protein-dependent structural changes and local interactions2014In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 107, no 2, p. 401-410Article in journal (Refereed)
    Abstract [en]

    Proteins function in cellular environments that are crowded with biomolecules, and in this reduced available space, their biophysical properties may differ from those observed in dilute solutions in vitro. Here, we investigated the effects of a synthetic macromolecular crowding agent, dextran 20, on the folded states of hyperthermophilic (S16T(herme)) and mesophilic (S161homologs of the ribosomal protein S16. As expected for an excluded-volume effect, the resistance of the mesophilic Meso, protein to heat-induced unfolding increased in the presence of dextran 20, and chemical denaturation experiments at different fixed temperatures showed the macromolecular crowding effect to be temperature-independent. Forster resonance energy transfer experiments show that intramolecular distances between an intrinsic Trp residue and BODIPY-labeled S16 Meso depend on the level of the crowding agent. The BODIPY group was attached at three specific positions in S16me, allowing measurements of three intraprotein distances. All S16meso variants exhibited a decrease in the average Trp-BODIPY distance at up to 100 mg/mL dextran 20, whereas the changes in distance became anisotropic (one distance increased, two distances decreased) at higher dextran concentrations. In contrast, the two 516-rhermo mutants did not show any changes in Trp-BODIPY distances upon increase of dextran 20 concentrations. It should be noted that the fluorescence quantum yields and lifetimes of BODIPY attached to the two S16 homologs decreased gradually in the presence of dextran 20. To investigate the origin of this decrease, we studied the BODIPY quantum yield in three protein variants in the presence of a tyrosine-labeled dextran. The experiments revealed distinct tyrosine quenching behaviors of BODIPY in the three variants, suggesting a dynamic local interaction between dextran and one particular S16 variant.

  • 34.
    Mikhalyov, I
    et al.
    Umeå University, Faculty of Science and Technology, Chemistry.
    Bogen, Stein-Tore
    Umeå University, Faculty of Science and Technology, Chemistry.
    Johansson, Lennart B-Å
    Umeå University, Faculty of Science and Technology, Chemistry.
    Donor-donor energy migration (DDEM) as a tool for studying aggregation in lipid phases2001In: SPECTROCHIMICA ACTA PART A-MOLECULAR AND BIOMOLECULAR, ISSN 1386-1425, Vol. 57, no 9, p. 1839-45Article in journal (Refereed)
    Abstract [en]

    A BODIPY(R)-labelled sulfatide (N-(BODIPY(R)- FL-pentanoyl) -galactosylcerebroside-sulfate, hereafter abbreviated as BD-Sulfatide) was solubilised at different concentrations in lipid vesicles of 1,2-dioleoyl-sn -glycero-3-phosphocholine (DOPC). Time-correlated single photon counting experiments show that the fluorescence relaxation is mono-exponential (with a lifetime of 6.5 ns) at molar ratios of BD-Sulfatide: DOPC that are less than 1:100. The fluorescence steady-state anisotropy decreases monotonously at molar ratios smaller than 1:1000, which is compatible with donor-donor energy migration (DDEM) among the BODIPY(R) groups. A model that assumes DDEM across the lipid bilayers, as well as in their planes, was used to analyse the time-resolved fluorescence anisotropy. Only two parameters appear in the model namely; the bilayer thickness (d) and the average number density (C-2) distribution of BD-Sulfatide in the lipid bilayers. The extracted d-values vary between 35 and 40 Angstrom, which is about the reported thickness of a bilayer of DOPC (38 Angstrom). Hence, the BODIPY(R) groups are preferentially located in the water-lipid interface. At low concentration the experimental C-2-values and those independently calculated are in good agreement, while the experimental values gradually become lower with increasing BD-Sulfatide concentration. These results are compatible with an aggregation of the sulfatides and self-quenching of BODIPY(R), which is clearly established at higher concentrations of the BD-Sulfatide.

  • 35.
    Mikhalyov, I
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Olofsson, Anders
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Gröbner, Gerhard
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Johansson, Lennart B-Å
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Designed fluorescent probes reveal interactions between Amyloid-β(1-40) Peptides and GM1 Gangliosides in Micelles and Lipid Vesicles2010In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 99, no 5, p. 1510-1519Article in journal (Refereed)
    Abstract [en]

    A hallmark of the common Alzheimer's disease (AD) is the pathological conversion of its amphiphatic amyloid-beta (Abeta) peptide into neurotoxic aggregates. In AD patients, these aggregates are often found to be tightly associated with neuronal G(M1) ganglioside lipids, suggesting an involvement of G(M1) not only in aggregate formation but also in neurotoxic events. Significant interactions were found between micelles made of newly synthesized fluorescent G(M1) gangliosides labeled in the polar headgroup or the hydrophobic chain and Abeta(1-40) peptide labeled with a BODIPY-FL-C1 fluorophore at positions 12 and 26, respectively. From an analysis of energy transfer between the different fluorescence labels and their location in the molecules, we were able to place the Abeta peptide inside G(M1) micelles, close to the hydrophobic-hydrophilic interface. Large unilamellar vesicles composed of a raftlike G(M1)/bSM/cholesterol lipid composition doped with labeled G(M1) at various positions also interact with labeled Abeta peptide tagged to amino acids 2 or 26. A faster energy transfer was observed from the Abeta peptide to bilayers doped with 581/591-BODIPY-C(11)-G(M1) in the nonpolar part of the lipid compared with 581/591-BODIPY-C(5)-G(M1) residing in the polar headgroup. These data are compatible with a clustering process of G(M1) molecules, an effect that not only increases the Abeta peptide affinity, but also causes a pronounced Abeta peptide penetration deeper into the lipid membrane; all these factors are potentially involved in Abeta peptide aggregate formation due to an altered ganglioside metabolism found in AD patients.

  • 36.
    Mikhalyov, Ilya
    et al.
    Umeå University, Faculty of Science and Technology, Chemistry.
    Gretskaya, Natalia
    Bergström, Fredrik
    Umeå University, Faculty of Science and Technology, Chemistry.
    Johansson, Lennart B-Å
    Umeå University, Faculty of Science and Technology, Chemistry.
    Electronic ground and excited state properties of dipyrrometheneboron difluoride (BODIPY): Dimers with application to biosciences2002In: PCCP Physical Chemistry, Chemical Physics and Biophysical Chemistry, Vol. 4, no 5663-70Article in journal (Refereed)
    Abstract [en]

    Numerous derivatives of BODIPY (4,4-difluoro-4-borata-3a-azonia-4a-aza-s-indacene) are frequently used as fluorescent probes in modern protein and lipid research. Present studies of purpose-synthesised molecules show that the BODIPY chromophore can form two different ground state dimers, denoted DI and DII. In practise DI exhibits negligible fluorescence emission, but a strong absorption band {(477 nm)=102000 mol–1 dm3 cm–1}, while DII is fluorescent with the radiative lifetime 20 ns, and red-shifted absorption {(577 nm)}=26000 mol–1 dm3 cm–1} relative to that of the monomer. Energy transfer is demonstrated from monomeric BODIPY to DI, as well as to DII. Donor–donor energy migration is also shown to occur between excited and ground state DII. Both DI and DII are of potential interest in examining structure-function of proteins and lipid membrane systems.

  • 37. Mikhalyov, Ilya
    et al.
    Gretskaya, Natalia
    Johansson, Lennart B-Å
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Fluorescent BODIPY-labelled GM1 gangliosides designed for exploring lipid membrane properties and specific membrane-target interactions2009In: Chemistry and Physics of Lipids, ISSN 0009-3084, E-ISSN 1873-2941, Vol. 159, no 1, p. 38-44Article in journal (Refereed)
    Abstract [en]

    New fluorophore-labelled GM1 gangliosides have been synthesised and spectroscopically characterised. Spectroscopically different BODIPY groups were covalently linked, specifically to either the polar or the hydrophobic part of the ganglioside molecule. The absorption and fluorescence spectroscopic properties are reported for 564/571-BODIPY- and 581/591-BODIPY-labelled GM1. Each of the different BODIPY groups is highly fluorescent and depolarisation experiments provide molecular information about the spatial distribution in lipid bilayers, as well as order and dynamics. From experiments performed on two spectroscopically different BODIPY:s, specific interactions can be revealed by monitoring the rate/efficiency of donor-acceptor electronic energy transfer. Systems of particular interest for applying these probes are e.g. mixtures of lipids, and peptides/proteins interacting with lipid membranes.

  • 38. Mukhtar, Emad
    et al.
    Bergström, Fredrik
    Umeå University, Faculty of Science and Technology, Chemistry.
    Johansson, Lennart B-Å
    Umeå University, Faculty of Science and Technology, Chemistry.
    Hyper Rayleigh Scattering Yields Improved Response Function in Analysing 2-Photon Excited Fluorescence2002In: Journal of Fluorescence, ISSN 1053-0509 (Print) 1573-4994 (Online), Vol. 12, no 3-4, p. 481-4Article in journal (Refereed)
    Abstract [en]

    An accurate instrumental response function is needed to conclusively deconvolute fluorescence data based on time-correlated single-photon counting (TCSPC) and multiphoton excitation. Routinely the response function is measured as Rayleigh scattering (RS) from a colloidal solution, even if the excitation is a multiphoton event. Present work demonstrates that a response function obtained as hyper Rayleigh scattering (HRS) provides a better choice for deconvolution of 2-photon excited fluorescence decays. The 1- and 2-photon response functions were monitored as RS and HRS from colloidal gold particles at 800 and 400 nm, respectively.

  • 39.
    Norlin, Nils
    et al.
    Umeå University, Faculty of Science and Technology, Chemistry.
    Håkansson, Per
    Umeå University, Faculty of Science and Technology, Chemistry.
    Westlund, Per-Olof
    Umeå University, Faculty of Science and Technology, Chemistry.
    Johansson, Lennart B-Å
    Umeå University, Faculty of Science and Technology, Chemistry.
    Extended Förster theory for determining intraprotein distances: Part III. Partial donor–donor energy migration among reorienting fluorophores2008In: Physical Chemistry Chemical Physics, Vol. 10, p. 6962-70Article in journal (Refereed)
    Abstract [en]

    An extended Förster theory (EFT) is derived and outlined for electronic energy migration between two fluorescent molecules which are chemically identical, but photophysically non-identical. These molecules exhibit identical absorption and fluorescence spectra, while their fluorescence lifetimes differ. The latter means that the excitation probability becomes irreversible. Unlike the case of equal lifetimes, which is often referred to as, donor–donor energy migration (DDEM), the observed fluorescence relaxation is then no longer invariant to the energy migration process. To distinguish, the present case is therefore referred to as partial donor–donor energy migration (PDDEM). The EFT of PPDEM is described by a stochastic master equation (SME), which has been derived from the stochastic Liouville equation (SLE) of motion. The SME accounts for the reorienting as well as the translational motions of the interacting chromophores. Synthetic fluorescence lifetime and depolarisation data that mimics time-correlated single photon counting experiments have been generated and re-analysed. The rates of reorientation, as well as the orientational configurations of the interacting D-groups were examined. Moreover the EFT of PPDEM overcomes the classical 2-problem and the frequently applied approximation of 2 = 2/3 in the data analyses. An outline for the analyses of fluorescence lifetime and depolarisation data is also given, which might prove applicable to structural studies of D-labelled macromolecules, e.g. proteins. The EFT presented here brings the analyses of PDDEM data to the same level of molecular detail as that used in ESR- and NMR-spectroscopy.

  • 40.
    Norlin, Nils
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Westlund, Per-Olof
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Johansson, Lennart B-Å
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Fluorescence spectroscopic properties analysed within the extended Förster theory with application to Biomacromolecular systems2009In: Journal of Fluorescence, ISSN 1053-0509, E-ISSN 1573-4994, Vol. 19, no 5, p. 837-845Article in journal (Refereed)
    Abstract [en]

    The extended Förster theory (EFT) of electronic energy transport accounts for translational and rotational dynamics, which are neglected by the classical Förster theory (FT). EFT has been developed for electronic energy transfer within donor-acceptor pairs [Isaksson, et al, Phys. 16 Chem. Chem. Phys., 9, 1941(2007)] and donor-donor pairs [Johansson, et al, J. Chem. Phys., 105, 10896 (1996); Norlin, et al, Phys. Chem. Chem. Phys., 10, 6962(2008)]. For donors that exhibit different or identical non-exponential fluorescence relaxation within a donor-donor pair, the process of reverberating energy migration is reversible to a higher or lower degree. Here the impact of the EFT has been studied with respect to its influence on fluorescence quantum yields, fluorescence lifetimes as well as depolarisation experiments. The FT predicts relative fluorescence quantum yields which usually agree with the EFT within experimental accuracy, however, substantial deviations occurs in the steady-state and in particular the time-resolved depolarisation data. 

  • 41.
    Olofsson, Maria
    et al.
    Umeå University, Faculty of Science and Technology, Chemistry.
    Kalinin, Stanislav
    Umeå University, Faculty of Science and Technology, Chemistry.
    Zdunek, Janusz
    Oliveberg, Mikael
    Umeå University, Faculty of Science and Technology, Chemistry.
    Johansson, Lennart B-Å
    Umeå University, Faculty of Science and Technology, Chemistry.
    Tryptophan-BODIPY: A versatile donor-acceptor pair for probing generic changes of intraprotein distances2006In: Physical Chemistry Chemical Physics, ISSN 1463-9076, Vol. 8, no 26, p. 3130-40Article in journal (Refereed)
    Abstract [en]

    We demonstrate that Tryptophan (Trp) and N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-yl)methyl iodoacetamide (BODIPY) is a suitable donor-acceptor (D-A) pair for intraprotein distance measurements, applicable to the study of protein folding. The suitability of the Trp-BODIPY electronic energy transfer is exemplified on the extensively-characterised two-state protein, S6, from Thermus thermophilus. This protein has proved to be useful for the elucidation of folding cooperativity and nucleation, as well as the changes upon induction of structural transitions. For a comprehensive structural coverage, BODIPY molecules were anchored by Cys insertions at four different positions on the S6 surface. Trp residues at position 33 or 62 acted as donors of electronic energy to the BODIPY groups. None of the D-A pairs show any detectable difference in the folding kinetics (or protein stability), which supports the notion that the two-state transition of S6 is a highly concerted process. Similar results are obtained for mutants affecting the N- and C-terminus. The kinetic analyses indicate that changes of the transition state occur through local unfolding of the native state, rather than by a decrease of the folding cooperativity. The distances obtained from the analysis of the time-resolved fluorescence experiments in the native state were compared to those calculated from X-ray structure. As an additional measure, molecular dynamics simulations of the different protein constructs were performed to account for variability in the BODIPY location on the protein surface. The agreement between fluorescence and X-ray data is quite convincing, and shows that energy transfer measurements between Trp and BODIPY can probe distances between ca. 17 to 34 A, with an error better than 10%.

  • 42.
    Opanasyuk, Oleg
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Johansson, Lennart B-Å
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Extended Förster theory: a quantitative approach to the determination of inter-chromophore distances in biomacromolecules2010In: Physical Chemistry, Chemical Physics - PCCP, ISSN 1463-9076, E-ISSN 1463-9084, Vol. 12, no 28, p. 7758-7767Article in journal (Refereed)
    Abstract [en]

    This review highlights recent theoretical and experimental advances in the study of biomacromolecular structure by using electronic transfer. The considered electronic transport in the extended Förster theory occurs within donor–acceptor pairs, donor–donor pairs, as well as within regular arrangements of many donors which may undergo reorienting and translational dynamics. The classical and the extended FoЁ rster theory are compared. Applications concern the determination of structural properties of proteins and non-covalent protein polymers. Studies of energy migration by means of two-photon excited fluorescence spectroscopy, as well as the relevant extension of the Förster theory are presented.

  • 43.
    Opanasyuk, Oleg
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Johansson, Lennart B-Å
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    On the analyses of fluorescence depolarisation data in the presence of electronic energy migration: Part I. Theory and general description2012In: Physical Chemistry, Chemical Physics - PCCP, ISSN 1463-9076, E-ISSN 1463-9084, Vol. 14, p. 1907-1916Article in journal (Refereed)
    Abstract [en]

    A new and general procedure is described for a detailed analysis of time-resolved fluorescence depolarisation data in the presence of electronic energy migration. An isotropic ensemble of bifluorophoric molecules (D1-R-D2) has been studied to demonstrate its utility. Intramolecular donor-donor energy migration occurs between the two donor groups (D), which are covalently connected to a rigid linker group (R). These groups undergo restricted reorientational motions with respect to the R group. The analysis of depolarisation data basically involves the search for best-fit parameters which describe the local reorienting motions, the intermolecular D1-D2 distance, as well as the mutual orientations of the donors. For this, the analysis is partly performed in the Fourier domain and the best-fit parameters are determined by using an approach based on a Genetic Algorithm. The energy migration process has been described by using Monte Carlo simulations and an extended Förster theory (EFT). It is found that the EFT provides the least time-consuming computational method. Since one-photon and two-photon excited fluorescence experiments can be applied for energy migration studies, a general and unified theoretical formulation is given.

  • 44.
    Opanasyuk, Oleg
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Mikaelsson, Therese
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Ryderfors, Linus
    Department of Photochemistry and Molecular Science, Uppsala University, P. O. Box 523, S-751 20 Uppsala, Sweden.
    Mukhtar, Emad
    Department of Photochemistry and Molecular Science, Uppsala University, P. O. Box 523, S-751 20 Uppsala, Sweden.
    Johansson, Lennart B-Å
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    On the analyses of fluorescence depolarisation data in the presence of electronic energy migration.: II. Applying & Evaluating Two-Photon Excited Fluorescence2012In: Physical Chemistry, Chemical Physics - PCCP, ISSN 1463-9076, E-ISSN 1463-9084, Vol. 14, p. 1917-1922Article in journal (Refereed)
    Abstract [en]

    Electronic energy migration within a bifluorophoric molecule has been studied by time-resolved two-photon excited (TPE) fluorescence depolarisation experiments. Data were analysed by using a recently developed quantitative approach [Opanasyuk, O. & Johansson, L. B.-Å., On the Analyses of Fluorescence Depolarisation Data in the Presence of Electronic Energy Migration. I. Theory & General Description. Phys. Chem. Chem. Phys., Submitted.]. The energy migration occurs between the 9-anthrylmethyl groups of the bifluorophoric molecule, bis-(9-anthrylmethylphosphonate) bisteroid. These groups undergo local reorientations, while overall tumbling of the bisteroid is strongly hampered in the used viscous solvent, 1,2-propanediol. To solely obtain information about local reorientations of the 9-anthrylmethyl group, also the mono-(9-anthrylmethylphosphonate) bisteroid was studied, which enabled modelling of the ordering potential shape. The analysis of data is partly performed in the Fourier domain and the best-fit parameters are determined by using an approach based on a Genetic Algorithm. The energy migration process was described by an extended Förster theory (EFT). A reasonable value of the distance between the 9-anthrylmethyl groups is found, as well as for the mutual orientation of the ordering potentials. Furthermore, values of the two-photon tensor components were obtained.

  • 45.
    Opanasyuk, Oleg
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Ryderfors, Linus
    Department of Photochemistry and Molecular Science, Uppsala University, P. O. Box 523, S-751 20 Uppsala, Sweden.
    Mukhtar, Emad
    Department of Photochemistry and Molecular Science, Uppsala University, P. O. Box 523, S-751 20 Uppsala, Sweden.
    Johansson, Lennart B-Å
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Two-photon excited fluorescence depolarisation and electronic energy migration within donor–donor pairs2009In: Physical Chemistry, Chemical Physics - PCCP, ISSN 1463-9076, E-ISSN 1463-9084, no 11, p. 7152-7160Article in journal (Refereed)
    Abstract [en]

    A unified theoretical description is presented for one- and two-photon excited fluorescence depolarisation and electronic energy migration within pairs of chromophores. Two weakly coupled donor groups are linked via a rigid macromolecule with the ability to undergo restricted reorienting motions. Describing these reorienting motions as well as their influence on the coupling is rather complex, but can be accounted for by using the extended Förster theory. Here explicit expressions have been derived for chromophores belonging to the point groups D2h,D2 andC2v when residing in uniaxial potentials (i.e. C∞v symmetry). From the given basic equations, it is possible however, to derive the relevant equations for molecules of arbitrary symmetry in any uniaxial orienting potential. The expected time-resolved fluorescence anisotropy for different two-photon absorption tensors are compared for reorienting fluorophores in liquids, as well as in anisotropic systems. Simulated fluorescence depolarisation data are also displayed that mimic energy migration within pairs of two-photon excited donor molecules, which simultaneously undergo reorienting motions within effectively isotropic and uniaxially anisotropic environments. The obtained results demonstrate that the time-resolved fluorescence anisotropy strongly depends on the properties of the two-photon absorption tensor, as well as on using a linear or a circular polarisation of the excitation field.

  • 46.
    Rosenbaum, Erik
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Sellstedt, Magnus
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Almqvist, Fredrik
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Johansson, Lennart B-Å
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Unusual light spectroscopic properties of a 2-Pyridone-based multi-ring-fused Fluorescent Scaffold2010In: Journal of Fluorescence, ISSN 1053-0509, E-ISSN 1573-4994, Vol. 20, no 6, p. 1249-53Article in journal (Refereed)
    Abstract [en]

    UV-VIS absorption and fluorescence spectroscopic properties of six related polyaromatic 2-pyridones have been studied. Excitation of the lowest and rather weak and structure-less transition [epsilon (max) (430 nm) approximately 3,000 mol-1dm3cm-1] gives rise to a broad fluorescence band in the visible region, for these compounds. These S0 <--> S1 transitions are compatible with symmetrically forbidden transitions, promoted by intensity borrowing, as is revealed by fluorescence depolarisation data. With one exception, all compounds exhibit strong fluorescence, with quantum yields in glycerol varying between 40% and 70%. The corresponding fluorescence lifetimes range from 11 ns to 17 ns, while the radiative lifetimes are very similar ( approximately 26 ns), for all compounds. Interestingly and rarely observed, the calculated radiative lifetimes for the weak absorption band are significantly longer, i.e. between 37 and 40 ns.

  • 47.
    Rosenbaum, Erik
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Tavelin, Staffan
    Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience, Clinical Pharmacology.
    Johansson, Lennart B-Å
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    A characterisation study on the application of inverted lyotropic phases for subcutaneous drug release2010In: International journal of pharmaceutics, ISSN 1873-3476, Vol. 388, no 1-2, p. 52-7Article in journal (Refereed)
    Abstract [en]

    An experimental characterisation of lipid mixtures consisting of inverted hexagonal and inverted cubic phases composed of soybean phosphatidylcholine (SPC) and glycerol dioleate (GDO) was performed. The release of five chromophores of varying lipophilicity, used as model drugs, was investigated. Two experimental setups were applied: one based on maintaining sink condition, while a constant volume release medium was employed for the other. For neither setup, no correlation between the model drug lipophilicity and the polarity of the carrier matrix was found. However, the lipid phases showed a prolonged release, spanning weeks, of the model drugs, which exhibit lipophilicity values ranging by four orders of magnitude.

  • 48. Ryderfors, Linus
    et al.
    Mukhtar, Emad
    Johansson, Lennart B-Å
    Umeå University, Faculty of Science and Technology, Chemistry.
    Excited-State Symmetry and Reorientation Dynamics of Perylenes in Liquid Solutions: Time-Resolved Fluorescence Depolarization Studies Using One- and Two-Photon Excitation2008In: The Journal of Physical Chemistry A, ISSN 1520-5215, Vol. 112, no 26, p. 5794-5803Article in journal (Refereed)
    Abstract [en]

    The excited-state symmetry and molecular reorientation of perylene, 1,7-diazaperylene, and 2,5,8,11-tetra-tert-butylperylene have been studied by different fluorescence depolarization experiments. The first excited electronic singlet state was reached through one-photon excitation (OPE) and two-photon excitation (TPE). A 400 and 800 nm femtosecond laser pulse was used for this purpose, and data were collected by means of the time-correlated single-photon counting technique. It is found that the rotational correlation times for each perylene derivative are very similar in the OPE and TPE depolarization experiments. For the determination of the two-photon absorption tensor, a recently described theoretical model has been applied (Ryderfors et al. J. Phys. Chem. A 2007, 111, 11531). It was found that the two-photon process can be described by a 2 × 2 absorption tensor for which the components are solvent dependent and exhibit mixed vibronic character. In the dipole approximation this is compatible with a parity-forbidden two-photon absorption into the first excited singlet state.

  • 49. Ryderfors, Linus
    et al.
    Mukhtar, Emad
    Johansson, Lennart B-Å
    Umeå University, Faculty of Science and Technology, Chemistry.
    The Symmetry of Two-Photon Excited States as Determined by Time-Resolved Fluorescence Depolarization Experiments2007In: The Journal of Physical Chemistry, Vol. 111, no 45, p. 11531-9Article in journal (Refereed)
    Abstract [en]

    A new experimental and theoretical approach is presented for the quantitative determination and assignment of the two-photon absorption tensor of fluorophores dissolved in liquid solutions. Two linearly independent time-resolved fluorescence anisotropies and the two-photon polarization ratio were determined from experiments based on using the time-correlated single photon counting technique. The data were analyzed in a global manner under the assumption of prevailing diffusive molecular reorientations and when accounting for the influence of rapid unresolved reorientations. The method has been applied in fluorescence studies of perylene, two-photon excited at 800 nm. The analysis suggests that the two-photon transition is mediated via vibronic coupling including at least two vibrations of different symmetry, and also that the first singlet excited electronic state acts as a dominating intermediate state.

  • 50. Ryderfors, Linus
    et al.
    Mukhtar, Emad
    Johansson, Lennart B-Å
    Umeå University, Faculty of Science and Technology, Chemistry.
    Two-Photon Excited Fluorescence and Molecular Reorientations in Liquid Solutions2007In: Journal of Fluorescence, ISSN 1053-0509 (Print) 1573-4994 (Online), Vol. 17, no 5, p. 466-80Article in journal (Refereed)
    Abstract [en]

    Theoretical expressions are derived that relate the two-photon excited fluorescence depolarisation experiments to the molecular symmetry and the rotational motions of fluorescent molecules. Diffusive rotational motions in liquid solvents are considered, as well as the influence of fast unresolved motions (e.g. librations). The results obtained are compared with one-photon excited fluorescence depolarisation experiments. The derived theoretical expressions can be applied for detailed analyses of the molecular rotation in solvent. Several of the results are useful for determining and assigning the components of two-photon absorption tensors.

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