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FOR2143


Institute of Physiology
University of Freiburg
Hermann-Herder-Str. 7
79104 Freiburg
Germany
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Physiology I
N.N.
Kommissarische Leitung:
Prof. Dr. Marlene Bartos

Physiology II
Prof. Dr. Bernd Fakler
Tel. +49 761 203-5176
Fax +49 761 203-5191
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  • We apply various configurations of the patch-clamp technique (whole-cell, cell-attached, excised inside-out, outside-out) to native and genetically manipulated cells and subcellular compartments. They enable us to monitor protein function and protein-protein interactions at high-resolution.
  • We use a large spectrum of biochemical techniques to detect and quantify membrane proteins (mainly ion channels and receptors), their post-translational modifications and association with other proteins in complexes and protein networks.
  • Modern mass spectrometers coupled with liquid chromatography enable us to identify several hundreds of proteins from complex samples with high confidence and sequence coverage. In addition, they provide quantitative data that let us determine stability, specificity and stoichiometry of protein-protein interactions as well as absolute protein abundance.
  • Nuclear magnetic resonance spectroscopy (NMR) provides information on structure and dynamics of biological macromolecules at atomic resolution under near-physiological conditions. We use it to examine proteins participating in the nano-environment of membrane proteins with regard to their 3D structure, mobility and interactions.
  • Using innovative microsystems, we work to enhance resolution and throughput of electrical recording of ionic currents. We develop biohybrid sensing devices based on single biological nanopores in membrane microarrays and study the interaction of natural and synthetic polymers with pore-forming membrane proteins.
  • To understand how neurons collectively process information, we develop optogenetic tools as well as new technologies for recordings from neurons in vivo and imaging of cell activity using photon Ca2+ and functional approaches. With computational network models we gain information on the principles underlying information processing in complex neuronal circuits.

Biological nanopores such as the protein alpha-hemolysin have become promising tools for label-free single-molecule analysis. Well-known examples include detection and characterization of polynucleotides or proteins as well as synthetic polyelectrolytes,1 single molecule force spectroscopy and single molecule mass spectrometry (reviewed in Ref. 2, 3) These measurements are performed by monitoring the block and unblock by single analyte molecules of the ionic conductance of a unitary pore reconstituted in a free-standing lipid bilayer. A more widespread use of protein nanopores with their many attractive applications in chemical and biological analytics will depend on the availability of recording methods that enable rapid collection of large amounts of data and can be easily automated. However, particularly for analytical tasks which require low noise and high frequency bandwidth recordings, low-capacitance microbilayers on smaller-than-standard  (e.g. < 100 µm) bilayers are required4-6 which are not readily produced in a microarray format. A current challenge is, therefore, to provide a high-density microarray platform for parallel bilayer recording without compromising on the precision of state-of-the-art single microbilayer nanopore experiments.

Using a novel technology platform developed together with the Laboratory for Chemistry and Physics of Interfaces at IMTEK (Professor Jürgen Rühe) we perform parallel high-resolution electrical single molecule analysis on a chip-based nanopore microarray. Lipid bilayers of <20 µm diameter containing single alpha-hemolysin pores are formed on arrays of sub-picoliter cavities containing individual microelectrodes (microelectrode cavity array, MECA) and ion conductance-based single molecule mass spectrometry is performed on mixtures of poly(ethylene glycol) molecules of different length. We can thereby demonstrate the function of the MECA device as a chip-based platform for array-format nanopore recordings with a resolution at least equal to that of established single microbilayer supports. Devices based on MECAs may enable more widespread analytical use of nanopores by providing the high throughput and ease of operation of a high-density array format while maintaining or exceeding the precision of state-of-the-art microbilayer recordings.

 

1. Murphy, R. J.; Muthukumar, M., Threading Synthetic Polyelectrolytes through Protein Pores. J. Chem. Phys. 2007, 126, 051101-,1-4
2. Kasianowicz, J. J.; Robertson, J. W. F.; Chan, E. R.; Reiner, J. E.; Stanford, V. M., Nanoscopic Porous Sensors. Annu. Rev. Anal. Chem. 2008, 1, 737-766.
3. Majd, S.; Yusko, E. C.; Billeh, Y. N.; Macrae, M. X.; Yang, J.; Mayer, M., Applications of Biological Pores in Nanomedicine, Sensing, and Nanoelectronics. Curr. Opin. Biotechnol. 2010, 21, 439-476.
4. Akeson, M.; Branton, D.; Kasianowicz, J. J.; Brandin, E.; Deamer, D. W., Microsecond Time-Scale Discrimination among Polycytidylic Acid, Polyadenylic Acid, and Polyuridylic Acid as Homopolymers or as Segments within Single Rna Molecules. Biophys. J. 1999, 77, 3227-3233.
5. Mayer, M.; Kriebel, J. K.; Tosteson, M. T.; Whitesides, G. M., Microfabricated Teflon Membranes for Low-Noise Recordings of Ion Channels in Planar Lipid Bilayers. Biophys. J. 2003, 85, 2684-2695.

6. Sondermann, M.; George, M.; Fertig, N.; Behrends, J. C., High-Resolution Electrophysiology on a Chip: Transient Dynamics of Alamethicin Channel Formation. Biochim. Biophys. Acta: Biomembranes 2006, 1758, 545-551.

 

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