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On the quantitative analysis of electronic energy transfer/migration in proteins studied by fluorescence spectroscopy
Umeå University, Faculty of Science and Technology, Chemistry.
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

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.

Place, publisher, year, edition, pages
Umeå: Kemi , 2007. , 46 p.
Keyword [en]
Biophysical Chemistry
National Category
Physical Chemistry
Identifiers
URN: urn:nbn:se:umu:diva-1009ISBN: 978-91-7264-263-8 (print)OAI: oai:DiVA.org:umu-1009DiVA: diva2:139967
Public defence
2007-03-02, KBC3A9, KBC-huset, 901 87, Umeå, 10:30 (English)
Opponent
Supervisors
Available from: 2007-02-15 Created: 2007-02-15 Last updated: 2009-10-28Bibliographically approved
List of papers
1. An environmental-sensitive BODIPY®-derivative with bioapplication: spectral and photophysical properties
Open this publication in new window or tab >>An environmental-sensitive BODIPY®-derivative with bioapplication: spectral and photophysical properties
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2003 (English)In: Journal of Fluorescence, ISSN 1053-0509 (Print) 1573-4994 (Online), Vol. 13, no 5, 379-84 p.Article in journal (Refereed) Published
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).

Keyword
BODIPY, donor–donor energy migration, homotransfer, dimers, quenching
Identifiers
urn:nbn:se:umu:diva-10060 (URN)doi:10.1023/A:1026156605013 (DOI)
Available from: 2008-06-12 Created: 2008-06-12 Last updated: 2009-10-23Bibliographically approved
2. Partial donor-donor energy migration (PDDEM): a novel fluorescence method for internal protein distance measurements
Open this publication in new window or tab >>Partial donor-donor energy migration (PDDEM): a novel fluorescence method for internal protein distance measurements
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2004 (English)In: Physical Chemistry, Chemical Physics - PCCP, ISSN 1463-9076, E-ISSN 1463-9084, Vol. 6, no 11, 3001-3008 p.Article in journal (Refereed) Published
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.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:umu:diva-14351 (URN)10.1039/b403264k (DOI)
Available from: 2007-06-26 Created: 2007-06-26 Last updated: 2017-12-14Bibliographically approved
3. Extended Förster theory for determining intraprotein distances: 1. The K2-dynamics and fluorophore reorientation
Open this publication in new window or tab >>Extended Förster theory for determining intraprotein distances: 1. The K2-dynamics and fluorophore reorientation
2004 (English)In: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 108, no 44, 17243-17250 p.Article in journal (Refereed) Published
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.

Keyword
Thimerosal, Ethylmercury, Mice, Autoimmunity
Identifiers
urn:nbn:se:umu:diva-2134 (URN)10.1021/jp040423d (DOI)
Available from: 2007-02-15 Created: 2007-02-15 Last updated: 2017-12-14Bibliographically approved
4. On the quantitative molecular analysis of electronic energy transfer within donor–acceptor pairs
Open this publication in new window or tab >>On the quantitative molecular analysis of electronic energy transfer within donor–acceptor pairs
2007 (English)In: Physical Chemistry, Chemical Physics - PCCP, ISSN 1463-9076, E-ISSN 1463-9084, Vol. 9, 1941-51 p.Article in journal (Refereed) Published
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.

Identifiers
urn:nbn:se:umu:diva-13009 (URN)doi:10.1039/b614817d (DOI)
Available from: 2007-06-26 Created: 2007-06-26 Last updated: 2017-12-14Bibliographically approved
5. Extentded Förster theory for determining intraprotein distances: 2. BODIPY-labelled plasminogen activator inhibitor type 1
Open this publication in new window or tab >>Extentded Förster theory for determining intraprotein distances: 2. BODIPY-labelled plasminogen activator inhibitor type 1
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Article in journal (Refereed) Submitted
Identifiers
urn:nbn:se:umu:diva-2136 (URN)
Available from: 2007-02-15 Created: 2007-02-15Bibliographically approved

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