Protonation dynamics, structure, and function of photoreceptors and cytochrome c oxidase (CcO)
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.
- Time-resolved fluorescence anisotropy and fluorescence pump-probe spectroscopy on the GPCR rhodopsin reveals that a conformational change of the amphiphatic helix 8 controls activation of arrestin
Kirchberg, K., Kim, T.-Y., Möller, M., Skegro, D., Dasara Raju, G., Granzin, J., Büldt, G., Schlesinger, R., and Alexiev, U. Proc. Natl. Acad. Sci. U S A. 108, 18690-5 (2011)
- Single molecule TIRF microscopy in combination with affinity fluorescence labeling via a G-protein derived peptide allows to determine the diffusivity of the active (metarhodopsin-II) visual receptor in the native disk membrane
- TIRF microscopy was used to develop a kinetic G-protein binding assay at the single molecule level
Kim, T.Y., Uji-i, H., Möller, M., Muls, B., Hofkens, J. and Alexiev, U., Biochemistry 48 (2009) 3801–3803)
Alexiev, U. and D. Farrens. Biochim. Biophys. Acta. 1837, 694-709 (2014)
- Site-directed fluorescence labeling of the terminal enzyme of the respiratory chain cytochrome c oxidase, an electron-coupled proton pump, in combination with simulation-guided fluorescence correlation spectroscopy (FCS) reveals precise local proton association and dissociation rates and provides information about protein surface effects at the entrance of the K channel. The absence of a dramatic increase in the protonation on- rate rules out a dramatic effect of a proton-collecting antenna for proton uptake through the K channel.
- Accelerated proton uptake was observed in the O-E step of the catalytic cycle of CcO by time-resolved flash-photolysis experiments
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)
- Site-directed fluorescence labeling of helix B in channelrhodopsin 2 and time-resolved anisotropy experiments reveal reveal that the formation of the open channel state is associated with a large conformational change at the cytoplasmic surface, consistent with an outward tilt of helix B. A pH-dependent structural heterogeneity was observed below pH 7. Knowledge about pH-dependent structural heterogeneity may be important for channelrhodopsin 2 applications in optogenetics.
Volz, P., Krause, N., Balke, J., Schneider, C., Walter, M., Schneider, F., Schlesinger, R. and Alexiev, U. J. Biol. Chem. 291, 33, 17382-17393 (2016)
- Cyanobacterial phytochromes are model systems for plant phytochromes. pH dependent analyses reveal a tight interplay between chromophore protonation state and protein assembly.
Escobar, F., Lang, C., Takiden, A., Schneider, C., Balke, J., Hughes, J., Alexiev, U., Hildebrandt, P. and Mroginski, M. J. Phys. Chem. B 121, 1, 47-57 (2017)
Nanomedicine: Analytical methods and visualization techniques for nanocarriers and targeted drug release
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
- Nanodynamics and unique fluorescence lifetimes of fluorescently tagged dendritic polyglycerols reveal temperature dependent cargo distribution in core multishell nanocarriers, size of nanocarrier systems, and cargo release
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)
- Single particle tracking (SPT) allows us to determine the diffusivity of drugs in skin and as cargo within loaded lipid nanoparticles. Superresolution microscopic approaches reveal the size and internal morphology of lipid nanoparticles
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)
- Fluorescence lifetime imaging microscopy allows for the discrimination of target molecules, e.g. fluorescently tagged nanocarriers, against the autofluorescent tissue background and, due to the environmental sensitivity of the fluorescence lifetime, also offers insights into the local environment of the nanoparticle and its interactions with other biomolecules.
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.
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.
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”