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Partial donor-donor energy migration (PDDEM): a novel fluorescence method for internal protein distance measurements
Umeå University, Faculty of Science and Technology, Department of Chemistry.
Umeå University, Faculty of Science and Technology, Department of Chemistry.
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
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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.

Place, publisher, year, edition, pages
2004. Vol. 6, no 11, 3001-3008 p.
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:umu:diva-14351DOI: 10.1039/b403264kOAI: oai:DiVA.org:umu-14351DiVA: diva2:154022
Available from: 2007-06-26 Created: 2007-06-26 Last updated: 2017-12-14Bibliographically approved
In thesis
1. On the quantitative analysis of electronic energy transfer/migration in proteins studied by fluorescence spectroscopy
Open this publication in new window or tab >>On the quantitative analysis of electronic energy transfer/migration in proteins studied by fluorescence spectroscopy
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
Biophysical Chemistry
National Category
Physical Chemistry
Identifiers
urn:nbn:se:umu:diva-1009 (URN)978-91-7264-263-8 (ISBN)
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
2. Electronic Energy Transfer within Asymmetric Pairs of Fluorophores: Partial Donor-Donor Energy Migration (PDDEM)
Open this publication in new window or tab >>Electronic Energy Transfer within Asymmetric Pairs of Fluorophores: Partial Donor-Donor Energy Migration (PDDEM)
2004 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

A kinetic model of electronic energy migration within pairs of photophysically non-identical fluorophores has been developed. The model applies to fluorescent groups that exhibit different photophysical and spectral properties when attached to different positions in a macromolecule. The energy migration within such asymmetric pairs is partially reversible, which leads to the case of partial donor-donor energy migration (PDDEM). The model of PDDEM is an extension of the recently developed donor-donor energy migration model (DDEM, F. Bergström et al, PNAS 96 (1999) 12477), and applies to quantitative measurements of energy migration rates and distances within macromolecules. One important distinction from the DDEM model is that the distances can be obtained from fluorescence lifetime measurements. A model of fluorescence depolarisation in the presence of PDDEM is also presented.

To experimentally test the PDDEM approach, different model systems were studied. The model was applied to measure distances between rhodamine and fluorescein groups within on-purpose synthesised molecules that were solubilised in lipid bilayers. Moreover, distances were measured between BODIPY groups in mutant forms of the plasminogen activator inhibitor of type 2 (PAI-2). Measurements of both the fluorescence intensity decays and the time-resolved depolarisation were performed. The obtained distances were in good agreement with independent determinations.

Finally, the PDDEM within pairs of donors is considered, for which both donors exhibit a nonexponential fluorescence decay. In this case it turns out that the fluorescence relaxation of a coupled system contains distance information even if the photophysics of the donors is identical. It is also demonstrated that the choice of relaxation model has a negligible effect on the obtained distances. The latter conclusion holds also for the case of donor-acceptor energy transfer.

Place, publisher, year, edition, pages
Umeå: Kemi, 2004. 36 p.
Keyword
Physical chemistry, fluorescence resonance energy transfer (FRET), donor-donor energy migration (DDEM), homotransfer, fluorescence relaxation, lifetimes, time-resolved fluorescence anisotropy, time-correlated single photon counting, distance measurements, protein structure, Fysikalisk kemi
National Category
Physical Chemistry
Research subject
Physical Chemistry
Identifiers
urn:nbn:se:umu:diva-338 (URN)91-7305-765-7 (ISBN)
Public defence
2004-11-19, KE 32, Kemihuset, Umeå University, Umeå, 13:00 (English)
Opponent
Supervisors
Available from: 2004-10-19 Created: 2004-10-19 Last updated: 2010-08-05Bibliographically approved
3. Plasminogen activator inhibitor type 2: a unique serpin with two mobile loops
Open this publication in new window or tab >>Plasminogen activator inhibitor type 2: a unique serpin with two mobile loops
2004 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The superfamily of serine protease inhibitors (serpins) is a large group of proteins with diverse functions but a common tertiary structure. Active serpins are highly metastable molecules. Metastability is the property underlying the success and ubiquitousness of serpins. However, serpin metastability also accounts for improper conformational changes in serpin mutants which may result in pathological serpin polymerization. Plasminogen activator inhibitor type 2 (PAI-2) is a member of the subfamily of ov-serpins. It is the only wild-type (wt) serpin that spontaneously polymerizes under physiological conditions. Another unique feature of PAI-2 is the loop connecting helices C and D (the CD-loop), which is the longest among known serpins and is involved in interactions with the environment.

Two partially overlapping goals were achieved during this thesis. The first was to study the molecular determinants involved in PAI-2 polymerization. By using in vitro mutagenesis in combination with biochemical and fluorescence methods, we have shown that wt PAI-2 exists both in stable monomeric and in polymerogenic conformations. The polymerogenic conformation can spontaneously form loop-sheet polymers and does not require conformational rearrangements prior to polymerization. The polymerogenic conformation of PAI-2 has an open β-sheet A, and it is stabilized by a disulfide bond formed between the unique CD-loop of PAI-2 and the bottom of the molecule. Under reducing conditions, the polymerogenic conformation of PAI-2 converts to the stable monomeric form. The polymerogenic and the stable monomeric forms are fully interconvertible, depending on the redox status of the environment. The stable monomeric conformation can be stabilized by vitronectin disulfide-bound to the CD-loop of PAI-2. The most populated conformation of the stable monomeric form of PAI-2 is that with the CD-loop folded on the side of the molecule. However, a mall fraction of stable monomeric PAI-2 can also exist with the CD-loop oriented toward the bottom of the molecule. Thus, the CD-loop of PAI-2 is a mobile molecular switch that regulates PAI-2 conformation.

The second goal of the thesis was to use PAI-2 as a model protein to develop methods for intramolecular distance measurements. An improved purification procedure, the stability of the protein and our understanding of its structure make PAI-2 an attractive candidate for use as a model protein. In this context, we have used PAI-2 successfully to measure distances by the previously used DDEM and newly developed PDDEM methods.

Place, publisher, year, edition, pages
Umeå: Medicinsk biokemi och biofysik, 2004. 61 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 870
Identifiers
urn:nbn:se:umu:diva-223 (URN)91-7305-572-7 (ISBN)
Public defence
2004-01-30, 00:00
Available from: 2004-03-30 Created: 2004-03-30 Last updated: 2010-08-05Bibliographically approved

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