The combination of fiber Bragg grating inscription with femtosecond laser sources and the usage of the Talbot interferometer setup not only gives access to the fabrication of Bragg gratings in new types of materials but also allows, at the same time, to keep the high flexibility of an interferometric setup in choosing the Bragg grating wavelength. Since the spatial and temporal coherence properties of the femtosecond laser source differ strongly from those of conventional laser sources, specific limits and tolerances in the interferometric setup have to be considered. Such limits are investigated on the basis of an analytical ray tracing model. The results are applied to tolerance measurements of fiber Bragg grating reflections recorded with a DUV sub-picosecond laser source at 262 nm. Additionally we demonstrate the wavelength versatility of the two-beam interferometer setup for femtosecond inscription over a 40 nm wavelength band. Inscription experiments in Al/Yb doped silica glasses are demonstrated as a prove for the access to non-photosensitive fibers.
The transmembrane (TM) helices of most type II single-span membrane proteins (S-SMPs) of Escherichia coli occur near the N-terminus, where the cell’s targeting mechanisms can readily identify it as it emerges from the ribosome. But the TM helices of a few S-SMPs, such as RodZ, occur a hundred or more residues downstream from the N-terminus, which raises fundamental questions about targeting and assembly. Because of RodZ’s novelty and potential usefulness for understanding TM helix insertion in vivo, we examined its membrane targeting and assembly. We used RodZ constructs containing immuno tags before the TM domain to assess membrane insertion using Proteinase K digestion. We confirmed the Nin-Cout (Type II) topology of RodZ and established the absence of a targeting signal other than the TM domain. RodZ was not inserted into the membrane under SecA-depletion conditions or in the presence of sodium azide, which is known to inhibit SecA. Insertion failed when the transmembrane proton gradient was abolished with CCCP. Insertion also failed when RodZ was expressed in SecE-depleted E. coli, indicating that the SecYEG translocon is required for RodZ assembly. Protease accessibility assays of RodZ in other E. coli depletion strains revealed that insertion is independent of SecB, YidC, and SecD/F. Insertion was found to be only weakly dependent on the signal recognition particle (SRP) pathway: insertion was weakly dependent on the Ffh but independent of FtsY. We conclude that membrane insertion of RodZ requires only the SecYEG translocon, the SecA ATPase motor, and the transmembrane proton motive force (PMF).
We report about a thermal regeneration of fiber Bragg gratings written in photosensitive fibers with nanosecond laser pulses. We observe a regenerative process in a highly photosensitive fiber without hydrogen loading which indicates a secondary grating growth in an optical fiber by thermal activation. This process is more temperature stable than the commonly known gratings produced by color center modifications. The writing conditions of such new type of gratings are investigated and the temperature behavior of these regenerated fiber Bragg gratings is analyzed. The application possibilities are in the field of high temperature sensor systems by making use of the combination of good spectral shape of a Type I grating with a Type II like temperature stability.
An oligo-leucine sequence has previously been shown to function as an artificial transmembrane segment that efficiently self-assembles in membranes and in detergent solution. Here, a novel technique, asparaginescanning mutagenesis, was applied to probe the interface of the self-assembled oligo-leucine domain. This novel approach identifies interfacial residues whose exchange to asparagine leads to enhanced self-interaction of transmembrane helices by interhelical hydrogen bond formation. As analyzed by the ToxR system in membranes, the interface formed by the oligo-leucine domain is based on a leucine-zipper-like heptad repeat pattern of amino acids. In general, the strongest impacts on self-assembly were seen with asparagines located around the center of the sequence, indicating that interaction is be more efficient here than at the termini of the transmembrane domains.
Under acid stress, Escherichia coli induce expression of CadA (lysine decarboxylase) and CadB (lysine/cadaverine antiporter) in a lysine-rich environment. The ToxR-like transcriptional activator CadC controls expression of the cadBA operon. Using a novel Signal Peptidase I (SPase I) cleavage assay, we show that CadC is a Type II single-span membrane protein (MP) with a cytoplasmic DNA binding domain and a periplasmic sensor domain. We further show that, as long assumed, dimerization of the sensor domain is required for activating the cadBA operon. We prove this using a chimera in which the periplasmic domain of RodZ—a Type II MP involved in the maintenance of the rod shape of E. coli—replaces the CadC sensor domain. Because the RodZ periplasmic domain cannot dimerize, the chimera cannot activate the operon. However, replacement of the TM domain of the chimera with the glycophorin-A (GpA) TM domain causes intramembrane dimerization and consequently operon activation. Using a low-expression protocol that eliminates extraneous TM-helix dimerization signals arising from protein over-expression, we enhanced dramatically the dynamic range of the β-galactosidase assay for cadBA activation. Consequently, the strength of the intramembrane dimerization of the GpA domain could be compared quantitatively with the strength of the much stronger periplasmic dimerization of CadC. For the signal-peptidase assay, we inserted a SPase I cleavage site (AAA or AQA) at the periplasmic end of the TM helix. Cleavage occured with high efficiency for all TM and periplasmic domains tested, thus eliminating the need for the cumbersome spheroplast-proteinase K method for topology determinations.
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