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On the analyses of fluorescence depolarisation data in the presence of electronic energy migration.: II. Applying & Evaluating Two-Photon Excited Fluorescence
Umeå University, Faculty of Science and Technology, Department of Chemistry. (Prof. Lennart B.-Å. Johansson)
Umeå University, Faculty of Science and Technology, Department of Chemistry. (Prof. Lennart B.-Å. Johansson)
Department of Photochemistry and Molecular Science, Uppsala University, P. O. Box 523, S-751 20 Uppsala, Sweden.
Department of Photochemistry and Molecular Science, Uppsala University, P. O. Box 523, S-751 20 Uppsala, Sweden.
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2012 (English)In: Physical Chemistry, Chemical Physics - PCCP, ISSN 1463-9076, E-ISSN 1463-9084, Vol. 14, 1917-1922 p.Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
2012. Vol. 14, 1917-1922 p.
Keyword [en]
Extended Förster theory, Genetic algorithms, Monte Carlo simulations, two-photon excitation
National Category
Physical Chemistry Atom and Molecular Physics and Optics
Research subject
Physical Chemistry
Identifiers
URN: urn:nbn:se:umu:diva-50049DOI: 10.1039/C2CP23177HOAI: oai:DiVA.org:umu-50049DiVA: diva2:458806
Available from: 2011-11-24 Created: 2011-11-24 Last updated: 2017-12-08Bibliographically approved
In thesis
1. A new approach to the analyses of fluorescence depolarisation experiments in the presence of electronic energy transport
Open this publication in new window or tab >>A new approach to the analyses of fluorescence depolarisation experiments in the presence of electronic energy transport
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
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 interfluorophore 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. It is found that this theory 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.

To exemplify the developed quantitative approach the depolarisation of the fluorescence in the presence of electronic energy migration within a bis-(9-anthrylmethylphosphonate) bisteroid molecule has been studied by time-resolved two-photon excited fluorescence depolarisation experiments. 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 of the donor. Values of the two-photon absorption tensor components were obtained. To describe the discrepancy between the measured values of the initial anisotropy and fundamental anisotropy predicted by theory the distribution of absorption tensor caused by fast processes have been introduced. An angular parameter of absorption tensor was determined. Reasonable values of the distance between the 9-anthrylmethyl groups, as well as for their mutual orientation were obtained.

Place, publisher, year, edition, pages
Umeå: Kemiska institutionen, Umeå University, 2011. 46 p.
Keyword
electronic energy transfer, donor-donor energy migration, extended Förster theory, fluorescence depolarisation, two-photon excitation, computer simulations, genetic algorithms
National Category
Physical Chemistry Atom and Molecular Physics and Optics
Research subject
Physical Chemistry
Identifiers
urn:nbn:se:umu:diva-50050 (URN)978-91-7459-331-0 (ISBN)
Public defence
2011-12-16, KBC-huset, KB3A9, "Lilla hörsalen", Umeå Universitet, Umeå, 10:00 (English)
Opponent
Supervisors
Available from: 2011-11-25 Created: 2011-11-24 Last updated: 2011-11-24Bibliographically approved
2. Electronic Energy Migration/Transfer as a Tool to Explore Biomacromolecular Structures
Open this publication in new window or tab >>Electronic Energy Migration/Transfer as a Tool to Explore Biomacromolecular Structures
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Fluorescence-based techniques are widely used in bioscience, offering a high sensitivity and versatility. In this work, fluorescence electronic energy migration/ transfer is applied to measure intramolecular distances in two types of systems and under various conditions.

The main part of the thesis utilizes the process of donor-acceptor energy transfer to probe distances within the ribosomal protein S16. Proteins are essential to all organisms. Therefore, it is of great interest to study protein structure and function in order to understand and prevent protein malfunction. Moreover, it is also important to try to study the proteins in an environment which resembles its natural habitat. Here two protein homologs were investigated; S16Thermo and S16Meso, isolated from a hyperthemophilic bacterium and a mesophilic bacterium, respectively. It was concluded that the chemically induced unfolded state ensemble of S16Thermo is more compact than the corresponding ensemble of S16Meso. This unfolded state compaction may be one reason for the increased thermal stability of S16Thermo as compared to S16Meso.

The unfolded state of S16 was also studied under highly crowded conditions, mimicking the environment found in cells. It appears that a high degree of crowding, induced by 200 mg/mL dextran 20, forces the unfolded state ensemble of S16Thermo to become even more compact. Further, intramolecular distances in the folded state of five S16 mutants were investigated upon increasing amounts of dextran 20. We found that the probed distances in S16Thermo are unaffected by increasing degree of crowding. However, S16Meso shows decreasing intramolecular distances for all three studied variants, up to 100 mg/mL dextran. At higher concentrations, the change in distance becomes anisotropic. This suggests that marginally stable proteins like s16Meso may respond to macromolecular crowding by fine-tuning its structure. More stable proteins like S16Thermo however, show no structural change upon increasing degree of crowding.

We also investigated the possibility of local specific interactions between the protein and crowding agent, by means of fluorescence quenching experiments. Upon increasing amounts of a tyrosine labelled dextran, a diverse pattern of fluorescence quantum yield and lifetime suggests that specific, local protein-crowder interactions may occur.

In a second studied system, electronic energy migration between two donor-groups, separated by a rigid steroid, was studied by two-photon excitation depolarization experiments. Data were analysed by using recent advances, based on the extended Förster theory, which yield a reasonable value of the distance between the two interacting donor-groups. To the best of our knowledge, this is the first quantitative analysis of energy migration data, obtained from two-photon excited fluorescence. 

Place, publisher, year, edition, pages
Umeå: Umeå Universitet, 2014. 55 p.
Keyword
Fluorescence, electronic energy transfer, two-photon excitation, small ribosomal protein S16, macromolecular crowding, dextran 20
National Category
Physical Chemistry
Research subject
Physical Chemistry
Identifiers
urn:nbn:se:umu:diva-86794 (URN)978-91-7601-021-1 (ISBN)
Public defence
2014-04-04, KBC-huset, KB3B1, Umeå universitet, Linnaeus Väg, Umeå, 09:00 (English)
Opponent
Supervisors
Funder
Swedish Research Council, 550021104
Available from: 2014-03-12 Created: 2014-03-11 Last updated: 2014-03-11Bibliographically approved

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