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Research project The project aims at a molecular-level-development of polarised light spectroscopy for insights into complex biostructures, in particular the dynamics, structure and functioning of proteins, as related to the folding and formation of regular protein aggregates (= non-covalent polymers). The studies of protein aggregation are related to fibrinolytic activity, amyloid diseases, actin polymerisation and pore-forming cytotoxins. In another branch of the project, enhanced fluorescence is studied for the development of amplifiers of chemical sensors, which can be used in e.g. point-of-care analysis.

For decades fluorescence resonance energy transfer (FRET) has been applied in studies of protein structure. From a physical point of view it is more enlightening to refer to the process as “resonance energy transfer” (RET) or “donor-acceptor energy transfer” (DAET), since the mechanism is a non-radiative process between electronic states. The application of RET on biosystems requires the specific insertion of a donor and an acceptor group into a structure, which turns out to be a fundamental limitation of the RET-method. However, this and other limitations of RET are circumvented by the work done within this project. It has been demonstrated that the use of two identical fluorescent groups solves the question of specificity, and also that chemically identical but photophysically non-identical groups can be used for obtaining quantitative distance information. Furthermore Förster's theory of RET is not valid for a detailed molecular analysis of experiments on most biomacromolecular systems. The extended Förster theory (EFT) developed within this project intends to bring the analyses of experiments to the same level of molecular description as in ESR and NMR spectroscopy. Hitherto this strive has been very promising.

Project overview

Project period:

2008-05-05 2010-12-31

Participating departments and units at Umeå University

Department of Chemistry, Faculty of Science and Technology

Research subject

Chemical sciences

Project description

1. Extended Förster theory and its further application. The EFT is the most detailed theory available for describing DDEM and DAET within pairs of interacting chromphores. A further development of the EFT also includes the case of partial DDEM, which is an ongoing collaboration with Prof. P.-O. Westlund (Department of Chemistry; Biophysical Chemistry, UmU). To theoretically describe the stochastic time-dependence of EFT one needs to generate orientational trajectories. In the data analysis the interacting probes are assumed to reorient in an effectively uniaxial potential, as modelled by a cone- or Maier-Saupe-potential. Another interesting aspect of the EFT is its application for the analyses of single-molecule fluorescence spectroscopic experiments. In a recently initialised project this is explored in collaboration with Prof. Claus Seidel and coworkers at the department of Physical Chemistry, Univ. of Düsseldorf, Germany. An important question concerns the reliability of molecular parameters determined from fitting the EFT to experimental data. For this the Levenberg-Marquardt algorithm is most commonly used, which searches the best fit in terms of statistical parameters, such as chi-square. Here the inherent uncertainty is whether the obtained parameters are unique. By using a very recent development, which is based on a Genetic Algorithm this limitation can be avoided. Here the parameter space is scanned by means of an ingenious algorithm, based on Darwin’s ideas of biological evolution.

2. Organisation of regular protein aggregates. Protein aggregation (confusingly named “protein polymerisation” in the biochemical and medical literature) raises several questions related to their
structure-dynamics-function. Intense fields of research concern the amyloid diseases (e. g. Creutzfeldt-Jakob´s disease, the Britain´s bovine spongiform encepathy, etc.), pore-forming proteins (cytotoxins), the formation and functioning of microtubulii and actin in cells. X-ray and NMR-techniques are not necessarily the ultimate choices, since the former technique relies on the cumbersome preparation of crystals of high quality, and the NMR experiments suffer from spectral resolution as the proteins become larger.

In the present form, the EFT is not applicable for theoretically describing the DAET, DDEM and PDDEM among more than two chromophores. However, a combination of Monte Carlo and Brownian dynamics simulations has been shown to work very well. Recently, this approach was applied to studies of BODIPY-labelled filamentous actin (F-actin) which revealed the known helical organization of F-actin demonstrating the usefulness of this technique for structural determination of complex protein aggregates. The distance from the filament axis to the fluorophore was found to be considerably less than expected from the proposed distance. A logical continuation of the F-actin studies is to label other positions in the protein, in order to reach new insights about the still unknown orientation of the actin subunit. These studies are currently running as part of a PhD-project in collaboration with Prof. Roger Karlsson at the Department of Cell Biology, SU, Prof. Uno Lindberg at KI and Prof. Clarence E Schutt, Princeton, USA.

The amyloid aggregates seem to generate cell death and perturb various cellular processes. It has been shown that aggregation of SERPIN:s (serine protease inhibitors) occurs in early Alzheimer´s disease causing liver as well as lung emphysema. Plasminogen activator inhibitor type 2 (PAI-2) is a SERPIN of interest in this context, because it forms aggregates under certain conditions. Moreover, the mechanism of PAI-2 aggregation is believed to be similar to that of the above-mentioned diseases. Knowledge of how PAI-2 forms aggregates may provide key-information to the understanding of a possible general mechanism for amyloid diseases. This project constitutes a new direction of an existing collaboration with Prof. Tor Ny and co-workers at the Department of Medical Chemistry, UmU.

3. Alzheimer peptide – ganglioside interactions. Lipid molecules, especially gangliosides, are thought to play an important role in binding of the Alzheimer peptide to lipid membranes, but also in the further formation of aggregates/fibrilles. Recently, it has been shown that the ganglioside GM1 exhibits a non-uniform lateral distribution within lipid bilayers. In order to detect the eventual affinity between these enriched regions of GM1 and the different mutant forms of the Alzheimer peptide, we are currently applying DDEM as well as DAET experiments. This project is running in collaboration with Dr. Ilya Mikhaylov (Laboratory of Lipid Research, Shemyakin & Ovchinnikov Institute, Moscow, Russia)and Docent Gerhard Gröbner at the Department of Chemistry; Biophysical Chemistry, UmU.

4. Enhanced fluorescence. The electronic properties of molecules located in the vicinity of nano-sized particles of Ag and Au may change dramatically, e.g. the fluorescence is theoretically predicted to increase by two orders of magnitudes36 depending on the shape of the particles. In a recent study a 20 times amplification was observed when using spherical Au particles. We have been studying the interaction between fluorophores and rod-shaped nano-Au particles of well-defined size, close to a year. The project is interesting from a basic science point of view, as well as for analytical applications. The latter aspect is investigated in collaboration with the Umeå Biotech Incubator.

5. Protein Folding. How proteins fold is a key question for interpreting the extensive information stored in the rapidly growing gene banks. The understanding of how genes are expressed at the level of protein structure appears to be a bottleneck for the development of modern biological research. At present one knows how genes are translated “letter by letter” into chains of amino acids, but one cannot predict the final structure of the corresponding proteins. From better knowledge of these rules, one might be able to understand, e.g. the origin of amyloid diseases, as well as predict to the structure and design of specific proteins which could have medicinal and industrial importance. The role of protein folding is a hot scientific enigma. To hopefully examine pathways of folding, we have discovered a versatile donor-acceptor pair by using a combination of Trp and BODIPY. This makes it possible to monitor the change in intramolecular distances upon perturbing a protein by denaturants, temperature and pressure. These studies relate to a project regarding the physico-chemical mechanisms of protein folding lead by Prof. Per-Olof Westlund (Department of Chemistry; Biophysical chemistry, UmU).

6. Two-photon excitation and fluorescence depolarisation. Knowledge about the polarisations of the involved electronic transition dipoles is a pre-requisite for any molecular interpretation of depolarisation data obtained by using one-photon excitation. The corresponding knowledge in the TPE depolarisation experiments concerns the polarisation of the electronic absorption transition tensor. The absorption tensors of most fluorescent molecules are not known, since they are theoretically and experimentally difficult to determine. These questions are examined in an on-going collaboration with Docent Emad Mukthar at the Ångström Laboratory in Uppsala.

7. DNA-sequencing. To distinguish between the four base pairs, we seek to develop four different fluorescent donor–acceptor pairs (so-called cassettes). In these cassettes a common D-group is used, which enables the use of a common excitation wavelength. Moreover, the rate of energy transfer should be efficient (preferably close to 100 %), and the A-groups should possess high quantum yields and separated fluorescence spectra. We have investigated different cassettes based on BODIPY as an acceptor group. This project is a continuing collaboration with Prof. Kevin Burgess and coworkers at the Department of Chemistry, Texas A & M University, USA.

8. Cataracts of the human lens. Throughout life the proteins of the lens undergo an increased turbidity and fluorescence, which correlates with high levels of blood sugar (diabetes), smoking, as well as exposure to sunlight; processes collectively named cataracts (Sw. starr). The coloured compounds are most likely formed through a non-enzymatic rearrangement of glucose derivatives. In an ongoing project, the coloured compounds are studied with respect to photostability and with the intention of finding conditions for a non-invasive photochemical decomposition of these compounds. Our studies show that the colouring and scattering of human lenses decrease upon exposure to certain wavelengths of UV-radiation45. These studies are performed in collaboration with Dr. Line Kessel and Prof. Michael Larsen (Københavns Amt, Amtsygehuset i Herlev, Øjenafdelingens forskningscenter, Danmark).

9. Bio-adapted fluorescent probes. This is a continuous search for newly and properly adapted fluorescent molecules, which is of interest for all the projects described above. There is a strive to develop small and highly fluorescent molecules. The development of probes is performed in collaborations with Prof. Heinz Langhals (Department of Organic Chemistry, University of Munich, Germany), Prof. Julian G. Molotkovsky, Dr. Ilya Mikhaylov (Shemyakin & Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russia and Prof. Kevin Burgess and at Department of Chemistry, Texas A & M University, USA.