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| Current research | ||||
| Counting membrane protein subunits with single molecule fluorescence | ||||
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Many receptors and ion channels are composed of several
subunits that have to co-assemble for proper protein function.
In the plasma membrane of the cell, these proteins form oligomers,
e.g. voltage-gated potassium channels form tetramers with four
voltage sensors around a central pore. In the lab of Ehud Isacoff, we developed a new optical method that is based on photobleaching of single molecules of the Green Fluorescent Protein (GFP) and is capable of resolving the oligomerization state of the protein in its natural environment, the cell membrane. We genetically fuse GFP to the protein of interest that we express at a very low density. Using Total Internal Reflection (TIR) Microscopy, we can observe how single GFP molecules loose their fluorescence during intense illumination due to destruction of the fluorophore. Observation of the GFP emission intensity allows to count the number of protein subunits by counting the steps until the intensity drops to zero. |
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| The subunit arrangement of novel voltage-sensing proteins | ||||
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Recently the genes for two new voltage-sensing proteins
were discovered: the voltage-sensor containing phosphatase
Ci-VSP (from Ciona intestinalis) and the voltage-dependent
proton channel Hv1. Their voltage sensor is assumed to be
similar to the voltage sensor of Shaker,
a voltage-dependent potassium channel, but it is lacking
the pore domain which forms the ion pathway in the
potassium channel, and at the same time serves as
the part that mediates the tetrameric assembly of the
Shaker channel. With our new method to count subunits of membrane protein subunits, we discovered that Ci-VSP is monomeric and Hv1 forms a dimer. We found that each subunit has its own pore by study of Hv1 tandem dimers that bear mutations in the part of the protein lining the conduction pathway. We also identified the dimerization interface of the Hv1 channel and constructed a chimeric protein from Hv1 and Ci-VSP that forms a monomer like Ci-VSP but features the proton conductance and the voltage dependence like Hv1. |
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| Previous research | ||||
| Bi-directional interfaces of nerve cells and micro-fabricated silicon devices | ||||
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The control of networks of neurons in vitro is an important step
towards understanding the principles of neuronal communication in the brain,
and a prerequisite for brain-machine interfacing. The choice of silicon as
a substrate offers the advantage of achieving a sensor density that is
comparable to the neuron density in the brain by employing modern silicon
processing techniques. Because there is no ionic or electronic current flow
across the silicon oxide surface, this approach is minimally invasive and
suitable for long term experiments. A bidirectional communication between silicon and cells requires field effect transistors and stimulation devices for sensing and eliciting action potentials in the cell. The bidirectional silicon-neuron interfaces developed by Peter Fromherz at the Max Planck Institute in Martinsried (Germany) helped to understand the physics of silicon-neuron coupling and boosted the sensor density for measuring extracellular field potentials to 16.384 transistors in an area of 1mm x 1mm. |
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