Fluorescence spectroscopy in combination with site-directed labelling has become an established tool to investigate the dynamics and interactions of biomolecules. The conformational flexibility and motion of membrane proteins are of particular interest as they play a key role in protein-protein interactions involved in function/activation, signaling protein interaction or receptor deactivation.
To understand the interplay between dynamics and function we combine protein dynamics investigations (via fluorescence) with kinetics experiments, such as flash photolysis experiments to track the photoreaction and protonation changes by time-resolved UV/Vis and fluorescence spectromicroscopy, fluorescence correlation spectroscopy (FCS), and fluorescence pump-probe measurements using multi-dimensional time-correlated single-photon counting. Single molecule TIRF experiments enable single particle tracking and the determination of diffusivities. Image-based methods have the advantage of tracking many particles at the same time allowing for parallelization.
Proteins investigated so far are visual rhodopsin (G-protein coupled receptor), channelrhodopsin (ion channel), phytochrome (photoreceptor), Cytochrome c oxidase (CcO), major histocompatibility complex (MHC) proteins.
Wolf, A., Schneider, C., Kim, T.Y., Kirchberg, K., Volz, P. and Alexiev, U. Phys. Chem. Chem. Phys.18, 12877-12885 (2016)
Kirchberg, K., Michel, H. and Alexiev, U. Biochim. Biophys. Acta. 1827, 276-84 (2013)
Kirchberg, K., Michel, H. and Alexiev, U. J. Biol. Chem., 287, 8187-93 (2012)
The mechanisms of drug-receptor interactions and the controlled delivery of drugs via biodegradable and biocompatible nanoparticulate carriers are active research fields in nanomedicine. In addition to the design of nano-sized multifunctional therapeutics and drug delivery systems the use and development of new analytical tools and devices are key elements of nanotechnology applied to nanomedicine.
Our research is focused on the skin penetration properties of macromolecular nanocarriers using single particle tracking (SPT), localization based superresolution microscopy, fluorescence resonance energy transfer (FRET), and fluorescence lifetime imaging (FLIM). Both molecular properties, such as dynamics of the nanocarriers, the influence on drug partitioning and targeted release, and cellular interactions of nanocarrier – drug systems are investigated. Advantages of local topical drug application and targeted drug delivery include the reduction of systemic undesirable side effects and the increase of local bioavailability
Boreham, A., Pfaff, M., Fleige, E., Haag, R., and Alexiev, U. Langmuir 30, 1686-95 (2014)
Boreham, A., Brodwolf, R., Pfaff, M., Kim, T.Y., Schlieter, T., Mundhenk, L., Gruber, A., Gröger, D., Licha, K., Haag, R. and Alexiev, U.. Polym. Adv. Technol. 25, 11, 1329-1336 (2014)
Boreham, A., Pikkemaat, J., Volz, P., Brodwolf, R., Kuehne, C., Licha, K., Haag, R., Dernedde, J. and Alexiev, U. Molecules 21(1), 22. (2016)
Boreham, A., Brodwolf, R., Walker, K., Haag, R. and Alexiev, U. Molecules 22 (1), 17, (2017)
Volz, P., Boreham, A., Wolf, A., Kim, T.Y., Balke, J., Frombach, J., Hadam, S., Afraz, Z., Rancan, F., Blume-Peytavi, U., Vogt, A. and Alexiev, U. International Journal of Molecular Sciences 16(4), 6960-6977 (2015)
Boreham, A., Volz, P., Peters, D., Keck, C. and Alexiev, U. Eur J Pharm Biopharm 110, 31-38 (2017)
Alnasif, N., Zoschke, C., Fleige, E., Brodwolf, R., Boreham, A., Rühl, E., Eckl, K.M., Merk, H.F., Hennies, H.C., Alexiev, U., Haag, R., Küchler, S. and Schäfer-Korting, M. J. Control. Release 185, 45-50 (2014)
Vogt, A., Wischke, C., Neffe, A.T., Ma, N., Müller, R.H., Alexiev, U. and Lendlein, A. J. Contr. Release. 242, 3-15, (2016)
Alexiev, U., Volz, P., Boreham, A., Brodwolf, R. Eur J Pharm Biopharm. (2017)
P Volz, P Schilrreff, R Brodwolf, C Wolff, J Stellmacher, J Balke, M Morilla, C Zoschke, M Schäfer-Korting, U Alexiev Ann. N.Y. Acad. Sci. 1405, 202-214, 2017.
Jens Balke, Pierre Volz, Falko Neumann, Robert Brodwolf, Alexander Wolf, Hannah Pischon, Moritz Radbruch, Lars Mundhenk, Achim D. Gruber, Nan Ma and Ulrike Alexiev, Small 14 (23), 18700106, 2018 doi: 10.1002/smll.201870106
New analytical methods and visualization techniques are key for both our research on nanocarriers and their tissue interaction and the function of membrane proteins and photoreceptors. We concentrate on the development of new analytical tools for SPT and FLIM measurements allowing us gain inside into the diffusion properties and localization and interaction of molecules in membranes, cells and tissue with an unprecedented accuracy.
- ps- and fs-time-resolved fluorescence / fluorescence depolarization
- Time correlated single photon counting (TCSPC)
- Pump-probe fluorescence spectroscopy
- 2-photon time-resolved fluorescence spectroscopy/microscopy
- Time-resolved absorption spectroscopy
- Single molecule total internal reflection fluorescence (TIRF) microscopy
- Fluorescence correlation spectroscopy (FCS)
Site-directed labeling has become an established tool to investigate the dynamics and interactions of biomolecules. We are using molecular biology tools to design and produce variants of CcO in the soil bacterium Paracoccus denitrificans. Biochemical methods are used to derivatize and characterize the proteins.
- Genetics laboratory (S1)
Project A2, PI U. Alexiev: Surface protonation and conformational dynamics in cytochrome c oxidase and photoreceptors
Integrated Graduate School (IGK), Speaker U.Alexiev: offers a structured program and support to all PhD students of the SFB during their doctoral studies.
Project B03, PI U. Alexiev: Characterization of transport and release properties of responsive nanocarriers by fluorescence spectroscopy and localization microscopy
HVI (Helmholtz Virtual Institute) “Multifunctional Biomaterials for Medicine”