Browsing by Author "Fischer, Daniela"

We report for the first time on pore-suspending lipid bilayers, which we call micro-black lipid membranes (micro-BLMs), based on a highly ordered macroporous silicon array. Micro-BLMs were established by first functionalizing the backside porous silicon surface with gold and then chemisorbing 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol followed by spreading 1,2-diphytanoyl-sn-glycero-3-phosphocholine dissolved in n-decane. Impedance spectroscopy revealed the formation of single lipid bilayers confirmed by a mean specific capacitance of 0.6 +/- 0.2 muF/cm(2). Membrane resistances were in the GOmega-regime and beyond. The potential of the system for single channel recordings was demonstrated by inserting the transmembrane domain of the HIV-1 accessory peptide Vpu(1-32), which forms helix bundles with characteristic opening states. We elucidated different amilorides as potential drugs to inhibit channel activity of Vpu.

Aggregation of the micro tubule-associated protein tau into neurofibrillary tangles is the pathological hallmark of a variety of dementias. For reasons not yet known, tau becomes excessively phosphorylated in Alzheimer's brains and as a result no longer binds properly to microtubules. Here we studied the impact of phosphorylation on the conformational and binding properties of the repeat region of tau (K18) that is necessary for microtubule assembly and forms the core of paired helical filaments. To mimic phosphorylation, we introduced four mutations of serine to glutamate residues at positions 262, 293, 324, and 356. NMR spectroscopy demonstrates that pseudophosphorylation at these sites modifies the structural properties in repeats 1 and 2, in particular for Gln265-Lys267. Gln265-Lys267 are in close proximity to Ser262, the phosphorylation site that most strongly attenuates binding to microtubules. In contrast, the pseudophosphorylation mimic of tau efficiently interacts with the polyanion heparin. Thus, phosphorylation of the repeat region of natively unfolded tau induces specific conformational changes that have a strong impact on its biological function and involvement. in disease.

Several mutations in the gene encoding the microtubule-associated protein tau are responsible for the formation of neurofibrillary inclusions in frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17). Here we present the high-resolution characterization of the conformational properties of two FTDP-17 mutants of the four-repeat domain of tau, P301L and Delta K280, and their properties for binding to polyanions and microtubules. Multidimensional NMR spectroscopy shows that the mutations do no lead to a significant increase in the level of beta-structure in their monomeric state, even though the mutations strongly promote beta-structure during aggregation. However, local structural changes are induced in the second repeat. These changes only weakly affect the binding to the polyanion heparin, which promotes paired helical filament formation. The extent of binding to microtubules, however, is strongly decreased. Our results demonstrate that the reversible binding of tau to microtubules involves specific interactions, which are not essential for binding to polyanions.

Tau is the major microtubule-associated protein in neuronal axons. It aggregates into "neurofibrillary tangles" during the course of Alzheimer disease. Binding to microtubules and microtubule assembly requires the "repeat domain" in the C-terminal half of Tau, as well as the two regions flanking the repeats. Here we report the NMR characterization of a 198-residue Tau fragment composed of the four tandem repeats and the flanking domains and containing the full microtubule binding and assembly activity of Tau. NMR secondary chemical shifts and dipolar couplings detect the highest propensity for beta-structure within the four-repeat region, whereas the flanking domains are largely random coil, with an increased rigidity in the proline-rich region. Chemical shift perturbation experiments identify two motifs in the upstream flanking domain, (225)KVAVVRT(231) and (243)LQTA(246), and one downstream of the repeats, (KIETHKTFREN380)-K-370, which strongly contribute to the binding to the acidic outside of microtubules, as well as to the binding of other polyanions such as heparin. This is consistent with the "jaws" model of Tau-microtubule interactions and highlights the importance of the regions flanking the repeats for both microtubule binding and pathological Tau aggregation.