Three-dimensional catheter tip force sensing using multi-core fiber Bragg gratings.

Awareness of catheter tip interaction forces is a crucial aspect during cardiac ablation procedures. The most important contact forces are the ones that originate between the catheter tip and the beating cardiac tissue. Clinical studies have shown that effective ablation occurs when contact forces are in the proximity of 0.2 N. Lower contact forces lead to ineffective ablation, while higher contact forces may result in complications such as cardiac perforation. Accurate and high resolution force sensing is therefore indispensable in such critical situations. Accordingly, this work presents the development of a unique and novel catheter tip force sensor utilizing a multi-core fiber with inscribed fiber Bragg gratings. A customizable helical compression spring is designed to serve as the flexural component relaying external forces to the multi-core fiber. The limited number of components, simple construction, and compact nature of the sensor makes it an appealing solution towards clinical translation. An elaborated approach is proposed for the design and dimensioning of the necessary sensor components. The approach also presents a unique method to decouple longitudinal and lateral force measurements. A force sensor prototype and a dedicated calibration setup are developed to experimentally validate the theoretical performance. Results show that the proposed force sensor exhibits 7.4 mN longitudinal resolution, 0.8 mN lateral resolution, 0.72 mN mean longitudinal error, 0.96 mN mean lateral error, a high repeatability, and excellent decoupling between longitudinal and lateral forces.

Topology of the SecA ATPase Bound to Large Unilamellar Vesicles.

The soluble cytoplasmic ATPase motor protein SecA powers protein transport across the Escherichia coli inner membrane via the SecYEG translocon. Although dimeric in solution, SecA associates monomerically with SecYEG during secretion according to several crystallographic and cryo-EM structural studies. The steps SecA follows from its dimeric cytoplasmic state to its active SecYEG monomeric state are largely unknown. We have previously shown that dimeric SecA in solution dissociates into monomers upon electrostatic binding to negatively charged lipid vesicles formed from E. coli lipids. Here we address the question of the disposition of SecA on the membrane prior to binding to membrane embedded SecYEG. We mutated to cysteine, one at a time, 25 surface-exposed residues of a Cys-free SecA. To each of these we covalently linked the polarity-sensitive fluorophore NBD whose intensity and fluorescence wavelength-shift change upon vesicle binding report on the the local membrane polarity. We established from these measurements the disposition of SecA bound to the membrane in the absence of SecYEG. Our results confirmed that SecA is anchored in the membrane interface primarily by the positive charges of the N terminus domain. But we found that a region of the nucleotide binding domain II is also important for binding. Both domains are rich in positively charged residues, consistent with electrostatic interactions playing the major role in membrane binding. Selective replacement of positively charged residues in these domains with alanine resulted in weaker binding to the membrane, which allowed us to quantitate the relative importance of the domains in stabilizing SecA on membranes. Fluorescence quenchers inside the vesicles had little effect on NBD fluorescence, indicating that SecA does not penetrate significantly across the membrane. Overall, the topology of SecA on the membrane is consistent with the conformation of SecA observed in crystallographic and cryo-EM structures of SecA-SecYEG complexes, suggesting that SecA can switch between the membrane-associated and the translocon-associated states without significant changes in conformation.

Stabilization of SecA ATPase by the primary cytoplasmic salt of Escherichia coli.

Much is known about the structure, function, and stability of the SecA motor ATPase that powers the secretion of periplasmic proteins across the inner membrane of Escherichia coli. Most studies of SecA are carried out in buffered sodium or potassium chloride salt solutions. However, the principal intracellular salt of E. coli is potassium glutamate (KGlu), which is known to stabilize folded proteins and protein-nucleic acid complexes. Here we report that KGlu stabilizes SecA, including its dimeric state, and increases its ATPase activity, suggesting that SecA is likely fully folded, stable, and active in vivo at 37°C. Furthermore, KGlu also stabilizes a precursor form of the secreted maltose-binding protein.

Effect of hydrogen gas on FBG-based optical fiber sensors for downhole pressure and temperature monitoring.

The influence of hydrogen gas on Fiber Bragg Grating (FBG)-based optical fiber sensors has been validated experimentally. More in particular, the focus was on FBGs written in the so-called Butterfly Micro Structured Fiber that targets simultaneous pressure and temperature monitoring with a minimum in cross-sensitivity to be used in, for example, downhole applications for the oil and gas market. The hydrogen-induced pressure and temperature errors from this type of sensor have been quantified as a function of the partial hydrogen pressure. The induced errors can be related to the diffusion of the hydrogen into the microstructure and to refractive index changes due to the presence of the hydrogen in the micro holes and penetration of it into the fiberglass. Furthermore, we have also shown that the hydrogen-induced errors scale with the partial hydrogen pressure.

Dropping Out and Other Fates of Transmembrane Segments Inserted by the SecA ATPase.

Type II single-span membrane proteins, such as CadC or RodZ, lacking a signal sequence and having a far-downstream hydrophobic segment, require the SecA secretion motor for insertion into the inner membrane of Escherichia coli. Using two chimeric single-span proteins containing a designed hydrophobic segment H, we have determined the requirements for SecA-mediated secretion, the molecular distinction between TM domains and signal peptides, and the propensity for hydrophobic H-segments to remain embedded within the bilayer after targeting. By means of engineered H-segments and a strategically placed SPase I cleavage site, we determined how targeting and stability of the chimeric proteins are affected by the length and hydrophobicity of the H-segment. Very hydrophobic segments (e.g., 16 Leu) are stably incorporated into the inner membrane, resulting in a C-terminal anchored membrane protein, while a 24L construct was not targeted to the membrane by SecA and remained in the cytoplasm. However, a construct carrying preMalE at the N-terminus led to SecA targeting to SecYEG via the native signal sequence and stable insertion of the downstream 24L H-segment. We show that the RseP intramembrane protease degrades weakly stable H-segments and is a useful tool for investigating the borderline between stable and unstable TM segments. Using RseP- cells, we find that moderately hydrophobic sequences (e.g., 5Leu + 11Ala) are targeted to SecYEG by SecA and inserted, but subsequently drop out of the membrane into the cytoplasm. Therefore, the free energy of transfer from translocon to bilayer is different from the transfer free energy from membrane to water.

FBGs written in specialty fiber for high pressure/high temperature measurement.

In this paper, we evaluate different thermal treatments in order to stabilize fiber Bragg gratings written by a femtosecond pulsed laser in specialty highly birefringent micro-structured optical fiber, targeting pressure monitoring at high pressure and high temperature environments. We have obtained a pressure sensitivity of 3.30 pm/bar up to 1400 bar and 290 °C. An effective thermal treatment has been experimentally implemented, yielding a nearly unchanged reflectivity at high temperature in combination with stable temperature and pressure readings: a standard deviation of 0.42 bar in the pressure reading was observed over 7 days at 280°C.

SecA drives transmembrane insertion of RodZ, an unusual single-span membrane protein.

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. However, 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 immunotags before the TM domain to assess membrane insertion using proteinase K digestion. We confirmed the N(in)-C(out) (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 TM proton gradient was abolished with Carbonyl cyanide m-chlorophenyl hydrazone. 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 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 TM proton motive force.

Topology, dimerization, and stability of the single-span membrane protein CadC.

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 (S-SMP) 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 membrane protein 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 transmembrane (TM) domain of the chimera with the glycophorin A 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 ß-galactosidase assay for cadBA activation. Consequently, the strength of the intramembrane dimerization of the glycophorin A domain could be compared quantitatively with the strength of the much stronger periplasmic dimerization of CadC. For the signal peptidase assay, we inserted an SPase I cleavage site (AAA or AQA) at the periplasmic end of the TM helix. Cleavage occurred with high efficiency for all TM and periplasmic domains tested, thus eliminating the need for the cumbersome spheroplast-proteinase K method for topology determinations.

Bending insensitivity of fiber Bragg gratings in suspended-core optical fibers.

This Letter presents simulation and experimental results that explore bending insensitivity of fiber Bragg gratings in suspended-core optical fibers. The implementation of thin silica bridge in the fibers enhances index contrast of the fiber core and reduces bending-induced strain transfer to the fiber core. This fiber design lead to a reduction of over 7 times in strain-induced fiber Bragg grating resonant peak shifts in the suspended-core fiber compared with that in standard telecommunication fiber, and an 0.14 dB bending loss at a bending radius of 6.35 mm.

Suspended-core fiber Bragg grating sensor for directional-dependent transverse stress monitoring.

This Letter presents simulation and experimental results of orientation-dependent transverse stress fiber sensors using fiber Bragg gratings (FBGs) inscribed in four-hole suspended-core fibers. Resonant peak shifts and splitting of FBGs were studied as functions of the applied transverse load and fiber orientation. Both simulation and experimental results revealed that the response of FBGs in suspended-core fibers is sensitive to both the orientation and magnitude of an applied transverse stress.

Post-hydrogen-loaded draw tower fiber Bragg gratings and their thermal regeneration.

The idea of Bragg gratings generated during the drawing process of a fiber dates back almost 20 years. The technical improvement of the draw tower grating (DTG) process today results in highly reliable and cost-effective Bragg gratings for versatile application in the optical fiber sensor market. Because of the single-pulse exposure of the fiber, the gratings behave typically like type I gratings with respect to their temperature stability. This means that such gratings only work up to temperatures of about 300 °C. To increase temperature stability, we combined DTG arrays with hydrogen postloading and a thermal regeneration process that enables their use in high-temperature environments. The regenerated draw tower gratings are demonstrated to be suitable for temperatures of more than 800 °C.

Thermal regeneration of fiber Bragg gratings in photosensitive fibers.

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.

Arrays of regenerated fiber bragg gratings in non-hydrogen-loaded photosensitive fibers for high-temperature sensor networks.

We report about the possibility of using regenerated fiber Bragg gratings generated in photosensitive fibers without applying hydrogen loading for high temperature sensor networks. We use a thermally induced regenerative process which leads to a secondary increase in grating reflectivity. This refractive index modification has shown to become more stable after the regeneration up to temperatures of 600 °C. With the use of an interferometric writing technique, it is possible also to generate arrays of regenerated fiber Bragg gratings for sensor networks.

Fiber Bragg grating inscription combining DUV sub-picosecond laser pulses and two-beam interferometry.

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.

An extended ToxR POSSYCCAT system for positive and negative selection of self-interacting transmembrane domains.

Assay systems based on the ToxR protein are widely used to investigate interaction of transmembrane domains that come from natural proteins or are isolated from combinatorial libraries. The principle of this method is that self-interaction of any given transmembrane domain, which is expressed within a ToxR chimeric protein, drives ToxR-ToxR assembly in a bacterial inner membrane. In current versions of the system, ToxR-ToxR interaction drives transcription activation of the cholera toxin (ctx) promoter and thereby induces expression of downstream reporter genes in appropriately constructed bacterial strains. Here, we describe the application of other known ToxR-regulated promoters. We show that interacting transmembrane domains also promote ToxR-driven activation of the ompU promoter. Conversely, these interactions efficiently repress transcription from the constitutively active ompT promoter. We present novel Escherichia coli strains whose chromosomes harbor fusions of ompU or ompT promoters with different reporter genes. Depending on the used promoter, self-interaction of transmembrane domains induces or represses reporter enzyme expression in these cells. These strains extend current applications of the ToxR protein and may find use in mapping transmembrane helix-helix interfaces and selection of transmembrane domains with medium affinities.

A ToxR-based dominant-negative system to investigate heterotypic transmembrane domain interactions.

The ToxR transcription activator system is a genetic tool widely used to examine homotypic interactions of alpha-helical transmembrane domains that drive or support oligomerization of integral membrane proteins. Here, we present a variant of this system that was developed to investigate heterotypic interactions. In this system, a homotypically interacting transmembrane domain within the ToxR protein is coexpressed with a potential competitor that is embedded within an inactivated ToxR mutant. Successful heterotypic interaction competes with homotypic interaction in a dominant-negative fashion as revealed by reduced reporter gene transcription. We expect that this system will be useful for the characterization of transmembrane domains whose homotypic and heterotypic interactions compete with each other.

The interface of a membrane-spanning leucine zipper mapped by asparagine-scanning mutagenesis.

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, asparagine-scanning 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.

Repair of hydrolytic DNA deamination damage in thermophilic bacteria: cloning and characterization of a Vsr endonuclease homolog from Bacillus stearothermophilus.

Hydrolytic deamination of 5-methyl cytosine in double stranded DNA results in formation of a T/G mismatch that-if left unrepaired-leads to a C-->T transition mutation in half of the progeny. In addition to several mismatch-specific glycosylases that have been found in both pro- and eukaryotes to channel this lesion into base excision repair by removing the T from the mismatch, Vsr endonuclease from Escherichia coli has been described which initiates repair by an endonucleolytic strand incision 5' to the mismatched T. We have isolated a gene coding for a homolog of E.coli Vsr endonuclease from the thermophilic bacterium Bacillus stearothermophilus H3 (Vsr.Bst) using a method that allows PCR amplification with degenerated primers of gene segments which code for only one highly conserved amino acid region. Vsr.Bst was produced heterologously in E.coli and purified to apparent homogeneity. Vsr.Bst specifically incises heteroduplex DNA with a preference for T/G mismatches. The selectivity of Vsr.Bst for the sequence context of the T/G mismatch appears less pronounced than for Vsr.Eco.

In vitro selection of self-interacting transmembrane segments--membrane proteins approached from a different perspective.

The principles underlying the folding of integral membrane proteins are uncovered in an increasingly detailed way. Experimental determination of high-resolution structures followed by analysis of packing reveal structural similarities as well as differences to soluble globular proteins. At the same time, protein/protein interactions at the level of membrane-embedded domains have been investigated for different model proteins. More recently, self-interacting transmembrane helices have been selected from combinatorial libraries in vitro to study the mechanistic basis of protein/protein interaction in membranes in a systematic way. With an emphasis on the latter approach, this review discusses insights emerging from an integrated view on the recent advances.