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- Håkansson, Pär, et al.
(author)
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Extended Förster theory for determining intraprotein distances : 1. The K2-dynamics and fluorophore reorientation
- 2004
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In: Journal of Physical Chemistry B. - : American Chemical Society (ACS). - 1520-6106 .- 1520-5207. ; 108:44, s. 17243-17250
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Journal article (peer-reviewed)abstract
- 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.
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- Isaksson, Mikael, et al.
(author)
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An environmental-sensitive BODIPY®-derivative with bioapplication : spectral and photophysical properties
- 2003
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In: Journal of Fluorescence. - 1053-0509 .- 1573-4994. ; 13:5, s. 379-84
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Journal article (peer-reviewed)abstract
- 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).
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- Isaksson, Mikael, et al.
(author)
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Extentded Förster theory for determining intraprotein distances : 2. An accurate analysis of fluorescence depolarisation experiments
- 2007
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In: Physical Chemistry, Chemical Physics - PCCP. - : Royal Society of Chemistry. - 1463-9076 .- 1463-9084. ; 9:29, s. 3914-3922
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Journal article (peer-reviewed)abstract
- 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 “κ2 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.
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- Isaksson, Mikael, 1974-
(author)
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On the quantitative analysis of electronic energy transfer/migration in proteins studied by fluorescence spectroscopy
- 2007
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Doctoral thesis (other academic/artistic)abstract
- Two recently developed theories of electronic energy transfer/migration were for the first time applied to real protein systems for extracting molecular distances. The partial donor-donor energy migration (PDDEM) is an extension to the previously developed donor-donor energy migration (DDEM, F Bergström et al PNAS 96, 1999, 12477) which allows using chemically identical but photophysically different fluorophores in energy migration experiments. A method based on fluorescence quenching was investigated and applied to create an asymmetric energy migration between fluorophores which were covalently and specifically attached to plasminogen activator inhibitor type 2 (PAI-2). It was also shown experimentally that distance information can be obtained if the fluorescence relaxation for photophysically identical donors, exhibits multi-exponential relaxation. An extended Förster theory (EFT) that was previously derived (L. B.-Å. Johansson et al J. Chem. Phys., 1996, 105) ha been developed for analysis of donor-acceptor energy transfer systems as well as DDEM systems. Recently the EFT was also applied to determine intra molecular distances in the protein plasminogen activator inhibitor type 1 (PAI-1) which was labelled with a sulfhydryl specific derivative of BODIPY. The EFT explicitly accounts for the time-dependent reorientations which in a complex manner influence the rate of electronic energy transfer/migration. This difficulty is related to the “k2-problem”, which has been solved. It is also shown experimentally that the time-correlated single-photon counting (TCSPC) data is sensitive to the mutual configuration between the interacting fluorophores. To increase the accuracy in the extracted parameters it is furthermore suggested to collect the fluorescence data under various physico-chemical conditions. It was also shown that the Förster theory is only valid in the initial part of the fluorescence decay.
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| 8. |
- Isaksson, Mikael, et al.
(author)
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On the quantitative molecular analysis of electronic energy transfer within donor–acceptor pairs
- 2007
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In: Physical Chemistry, Chemical Physics - PCCP. - : Royal Society of Chemistry (RSC). - 1463-9076 .- 1463-9084. ; 9, s. 1941-51
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Journal article (peer-reviewed)abstract
- 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.
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| 9. |
- Isaksson, Mikael, et al.
(author)
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Partial donor-donor energy migration (PDDEM) : a novel fluorescence method for internal protein distance measurements
- 2004
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In: Physical Chemistry, Chemical Physics - PCCP. - : Royal Society of Chemistry (RSC). - 1463-9076 .- 1463-9084. ; 6:11, s. 3001-3008
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Journal article (peer-reviewed)abstract
- 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.
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| 10. |
- Mulvenna, Maurice, et al.
(author)
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Towards sustainable business models from technology healthcare research
- 2010
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In: International Journal of Computers in Healthcare (IJCIH). - Geneve : Inderscience Enterprises Lilmited. - 1755-3199. ; 1:1, s. 43-51
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Journal article (peer-reviewed)abstract
- As demographic ageing impacts across the world, health and welfare organisations are seeking new paradigms of care that address people’s needs as well as being inherently more scalable than the incumbent processes and services. The aim of this paper is to describe the current situation in Europe with information on service provision, before signposting some possible new ways to develop sustainable business models that support care models. The paper uses a case study approach to examine the issues in the introduction of such business models, from a perspective of the translation of research proof of concepts into business services and from the perspective of developing innovations from research that address unmet or poorly considered needs of user. The paper shows how several innovative European projects are anticipating the need for service change and are designing their research outcomes to match the needs of service commissioners more fully. The conclusion discusses several different approaches before drawing together trands of the work and providing tentative recommendations on the way forward to develop new inclusive technology-enhanced services in health and social care.
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