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Current re­search undertaken in Kasan­mascheff Lab revolves around understanding the in vitro and in vivo structure, function and dynamics of biomolecules and bioorganic radicals that play key roles in life such as biosynthesis of DNA building blocks, higher-order DNA G-quadruplex structures, FeS proteins and proton-coupled electron transfer reactions. To reach our goals, we extensively employ EPR spectroscopy combined with sophisticated biochemical and biophysical techniques.

In vivo structure determination of biomolecules

The function of biomolecules is dictated by their structure and dynamics, which are affected by the protein environment. In vitro experiments might differ from the in vivo ones where molecular crowding, protein-protein interactions and the concentration of co-solutes may play significant roles on the structure and conformational heterogeneity of proteins. Therefore, in our lab we study biomolecules in their natural environment, the biological cell.

Our first target is the E. coli ribonucleotide reductase (RNR) that catalyzes the biosynthesis of DNA building blocks in E. coli and humans. RNRs are a paradigm to study radical transfer steps in essential protein machineries and also well-recognized targets for cancer therapeutics. Therefore, determining its in vivo structure and regulation is of high significance (see re­search highlights for more detail).

Figure 1: Different EPR techniques are employed to give insights into in vivo structure and function of E. coli ribonucleotide reductase.

Higher-order DNA G-quadruplex structures and their formation kinetics

G-qua­dru­plexes are DNA secondary structures containing stacked guanine tetrads stabilized by central cations. Studies have shown that G-qua­dru­plexes form in vivo in oncogene regulatory regions and at telomeric ends of chromosomes, thus shortening cancer cell lifetimes. Many G-quadruplex species are known to form higher-order structures like dimers, which are believed to play a crucial role in G-qua­dru­plexes biological activity. This makes the understanding of the formation of these structures an im­por­tant goal. We use EPR spectroscopy to gain insight into the topology of these higher-order structures by deriving CuII–CuII distances in various forms of G-quadruplex dimers, in which Cu(pyridine)4 complexes are covalently incorporated into tetramolecular G-qua­dru­plexes. With the CuII-based spin labels attached at either 3’ or 5’ ends of several G-quadruplex species, we show the dimerization at the opposite ends and follow the kinetics of heterogenous dimerization via dipolar spectroscopy.

Figure 2: A) Schematic representation of DNA G-quadruplex dimers (right). Orientation selective distance measurements with background-corrected orientation-averaged distance measurement (upper left) and corresponding distance distribution (lower left). B) Schematic representation of sandwich complex of DNA G-quadruplex and free guanine tetrad (left). Orientation selective distance measurements with background-corrected orientation-averaged distance measurement (upper right) and corresponding distance distributions (lower right). The two distances represent the DNA G-quadruplex dimers and the sandwich complex.

EPR-monitored redox potentiometry

Determination of redox potentials of FeS proteins provide essential in­for­mation on the energetics of biological electron transfer reactions. Therefore, we built an electrochemical cell for mediated titrations of metalloproteins, and thus obtaining their redox potentials via EPR-monitored potentiometric titrations. We established a protocol for the accurate determination of the redox potentials via pulse EPR for the first time. We started determining the redox potentials of ferredoxins (Fdxs), which are small, soluble FeS proteins. Additionally, we develop strategies to tune the midpoint potentials of selected ferredoxin, i.e., point mutations of charged residues on the protein surface and/or in vicinity of the FeS clusters.

Figure 3: (A) Electron-spin echo EPR spectra of different plant-type Fdx isoforms and their corresponding simulations. Plots of normalized spin concentrations against the respective potentials of the reductive (circles) and oxidative (squares) redox potentiometries of PetF (Fdx1) and Fdx2, Fdx3 and Fdx7.

Light-activated radicals

Tyrosine (Y) and tryptophan (W) residues can serve as one-electron redox cofactors in biocatalysis and multistep proton-coupled electron transfer (PCET) reactions. Enzymes that employ amino-acid radical cofactors are involved in essential processes in primary metabolism such as photosynthesis, respiration, and biosynthesis of DNA building blocks. Therefore, detailed understanding of amino-acid radical mediated PCET reactions is of vital interest. In order to investigate the amino-acid radical meditated PCET, we utilize light activation and EPR spectroscopy on two distinct systems: a model protein that is specifically designed to study amino-acid radicals, and a synthetic system that mimics the tyrosine dyad in RNR (DPX).

Figure 4: Schematic representation of an amino-acid radical and DPX (B).


Location & approach

The campus of TU Dort­mund Uni­ver­sity is located close to interstate junction Dort­mund West, where the Sauerlandlinie A 45 (Frankfurt-Dort­mund) crosses the Ruhrschnellweg B 1 / A 40. The best interstate exit to take from A 45 is "Dort­mund-Eichlinghofen" (closer to Campus Süd), and from B 1 / A 40 "Dort­mund-Dorstfeld" (closer to Campus Nord). Signs for the uni­ver­si­ty are located at both exits. Also, there is a new exit before you pass over the B 1-bridge leading into Dort­mund.

To get from Campus Nord to Campus Süd by car, there is the connection via Vo­gel­pothsweg/Baroper Straße. We recommend you leave your car on one of the parking lots at Campus Nord and use the H-Bahn (suspended monorail system), which conveniently connects the two campuses.

TU Dort­mund Uni­ver­sity has its own train station ("Dort­mund Uni­ver­si­tät"). From there, suburban trains (S-Bahn) leave for Dort­mund main station ("Dort­mund Hauptbahnhof") and Düsseldorf main station via the "Düsseldorf Airport Train Station" (take S-Bahn number 1, which leaves every 20 or 30 minutes). The uni­ver­si­ty is easily reached from Bochum, Essen, Mülheim an der Ruhr and Duisburg.

You can also take the bus or subway train from Dort­mund city to the uni­ver­si­ty: From Dort­mund main station, you can take any train bound for the Station "Stadtgarten", usually lines U41, U45, U 47 and U49. At "Stadtgarten" you switch trains and get on line U42 towards "Hombruch". Look out for the Station "An der Palmweide". From the bus stop just across the road, busses bound for TU Dort­mund Uni­ver­sity leave every ten minutes (445, 447 and 462). Another option is to take the subway routes U41, U45, U47 and U49 from Dort­mund main station to the stop "Dort­mund Kampstraße". From there, take U43 or U44 to the stop "Dort­mund Wittener Straße". Switch to bus line 447 and get off at "Dort­mund Uni­ver­si­tät S".

The H-Bahn is one of the hallmarks of TU Dort­mund Uni­ver­sity. There are two stations on Campus Nord. One ("Dort­mund Uni­ver­si­tät S") is directly located at the suburban train stop, which connects the uni­ver­si­ty directly with the city of Dort­mund and the rest of the Ruhr Area. Also from this station, there are connections to the "Technologiepark" and (via Campus Süd) Eichlinghofen. The other station is located at the dining hall at Campus Nord and offers a direct connection to Campus Süd every five minutes.

The AirportExpress is a fast and convenient means of transport from Dort­mund Airport (DTM) to Dort­mund Central Station, taking you there in little more than 20 minutes. From Dort­mund Central Station, you can continue to the uni­ver­si­ty campus by interurban railway (S-Bahn). A larger range of in­ter­na­tio­nal flight connections is offered at Düsseldorf Airport (DUS), which is about 60 kilometres away and can be directly reached by S-Bahn from the uni­ver­si­ty station.

The facilities of TU Dort­mund Uni­ver­sity are spread over two campuses, the larger Campus North and the smaller Campus South. Additionally, some areas of the uni­ver­si­ty are located in the adjacent "Technologiepark".

Site Map of TU Dort­mund Uni­ver­sity (Second Page in English).