Involvement of a conserved GFG motif region in substrate binding by RseP, an Escherichia coli S2P protease.

RseP, an Escherichia coli S2P family intramembrane cleaving protease, is involved in regulation of the extracytoplasmic stress response and membrane quality control through specific cleavage of substrates. Recent research suggested that the PDZ domains and the MRE ß-loop (membrane-reentrant ß-loop) are involved in substrate discrimination; the former would serve to prevent cleavage of substrates with a large periplasmic domain, whereas the latter would directly interact with the substrate's transmembrane segment and induce its conformational change. However, the mechanisms underlying specific substrate recognition and cleavage by RseP are not fully understood. Here, the roles of the N-terminal part of the first cytoplasmic loop region (C1N) of RseP that contains a highly conserved GFG motif were investigated. A Cys modifiability assay suggested that C1N is partly membrane-inserted like the MRE ß-loop. Pro, but not Cys, substitutions in the GFG motif region compromised the proteolytic function of RseP, suggesting the importance of a higher order structure of this motif region. Several lines of evidence indicated that the GFG motif region directly interacts with the substrate and also aids the function of the MRE ß-loop that participates in substrate recognition by RseP. These findings provide insights into the substrate recognition mechanisms of S2P proteases.

Achromatic prism-type wave plate for broadband terahertz pulses.

We demonstrated achromatic half- and quarter-wave plates for broadband terahertz pulses using phase retardation by internal total reflection. Prism-type wave plates realized ultra-broadband retardation stability up to 2.5 THz, which was the limitation of our experimental setup. Novel aspects of our work were use of a 3λ/4 plate as a quarter-wave plate and a multistacked prism-type (MSP) wave plate for a large-aperture THz beam. Real-time polarization imaging of two crossed bunches of hairs was performed to show the efficiency of the MSP wave plate. We clearly observed polarization dependence of the hair direction.

Single-shot terahertz spectroscopy using pulse-front tilting of an ultra-short probe pulse.

We developed a single-shot terahertz pulse measurement technique using pulse-front tilting of an ultra-short probe pulse and demonstrated single-shot terahertz spectroscopy. A transmission grating was used to introduce a sufficiently large pulse-front tilt angle. A measuring time range of 23.8 ps was achieved. The measured temporal waveforms were corrected in consideration of the nonlinearity arising from the crossed-Nicols arrangement employed and the beam profiles of the probe and terahertz pulses. The characteristic spectrum of lactose was measured with a single terahertz pulse, and the effectiveness of our single-shot technique was confirmed by comparison with a conventional sampling method.

Spectral imaging of pharmaceutical materials with a compact terahertz difference-frequency generation semiconductor source.

Spectral imaging of pharmaceutical material using a compact ultra-broadband (1-4 THz) terahertz semiconductor source was demonstrated. False-color RGB images could be obtained using a simple procedure (calibration free). The ability to distinguish the polymorphism of carbamazepine (CBZ), the hydrate forms of D-(+)-glucose and caffeine, and the crystallinity of nifedipine was demonstrated using the THz DFG source. Crystal forms of pharmaceutical materials can be distinguished using this method.

In Vivo and In Vitro Assay for Monitoring the Topological Loading of Bacterial Condensins on DNA.

Condensins play essential roles in the compaction and segregation of chromosomal DNA in life forms ranging from bacteria to higher organisms. To elucidate the molecular mechanisms underlying these roles, it is crucial to determine how and where condensins are loaded to chromosomal DNA. Here, we describe in vivo and in vitro assays for monitoring the topological loading of two bacterial condensins, Smc-ScpAB and MukBEF. A key step in these assays is washing the samples with a high concentration of salt in order to discriminate between electrostatic and topological binding of the bacterial condensins to DNA. In addition, isolation of bacterial condensin and DNA complexes prevents any undesired interaction between them due to cross-linking reagents. These methodologies provide reproducible and reliable results for the loading of topologically bound proteins such as bacterial condensins.

Pulsed Terahertz Radiation for Sensitive Quantification of Carbonate Minerals.

Quantification of natural carbonate minerals, namely, aragonite, high- and low-Mg calcite, and dolomite provides essential information about biomineralization, carbon cycling on Earth, and the evolution of ocean chemistry, and is also useful in many other scientific, pharmaceutical, and industrial fields. However, X-ray diffractometer has previously been the only practical tool to identify and quantify carbonate minerals, including calcium carbonate (CaCO3) polymorphs. We propose new fingerprint terahertz (THz) absorption and reflective index spectra in the 1-6 THz range that probe the lattice phonon modes and can be used for sensitive quantification of these four carbonate minerals, including polymorphs. In THz time-domain spectroscopy with our unique attenuated total reflection system, high- and low-Mg calcite and aragonite show different absorbance and reflective index amplitudes at 3.32 THz, which corresponds to the transverse optic mode. Dolomite shows a distinct absorbance peak and reflective index at 4.82 THz because its space group (R3̅) is different from that of calcite (R3̅c). THz absorbance and reflective index curves of the mixed carbonate materials, which typically occur in natural environments, correspond well to the curves calculated from the results of single-mineral samples (R 2 > 0.98). Remarkably, the absorbance and reflective index can quantify small fractions (<1%) of low-Mg calcite in an aragonite matrix with high linearity (R 2 = 0.99). Our findings provide a new method for screening low-Mg calcite diagenetic overprints on primary aragonitic skeletons such as corals, which is crucial for climate reconstructions using the isotopic analyses because a 1% overprint can cause estimated temperature deviations of ∼1 °C. THz spectra of carbonate minerals offer not only a new high-sensitivity quantification tool for interdisciplinary fields, but also safer light-source handling than X-ray diffractometer.

Roles of the membrane-reentrant ß-hairpin-like loop of RseP protease in selective substrate cleavage.

Molecular mechanisms underlying substrate recognition and cleavage by Escherichia coli RseP, which belongs to S2P family of intramembrane-cleaving proteases, remain unclear. We examined the function of a conserved region looped into the membrane domain of RseP to form a ß-hairpin-like structure near its active site in substrate recognition and cleavage. We observed that mutations disturbing the possible ß-strand conformation of the loop impaired RseP proteolytic activity and that some of these mutations resulted in the differential cleavage of different substrates. Co-immunoprecipitation and crosslinking experiments suggest that the loop directly interacts with the transmembrane segments of substrates. Helix-destabilising mutations in the transmembrane segments of substrates suppressed the effect of loop mutations in an allele-specific manner. These results suggest that the loop promotes substrate cleavage by selectively recognising the transmembrane segments of substrates in an extended conformation and by presenting them to the proteolytic active site, which contributes to substrate discrimination.

Evaluation of drug crystallinity in aqueous suspension using terahertz time-domain attenuated total reflection spectroscopy.

Terahertz pulsed spectroscopy has recently been demonstrated to be a novel technique for the investigation of the solid-state properties of pharmaceutical materials. In this study, we directly measured the crystallinity of a drug suspended in water, using a terahertz pulsed attenuated total reflection (ATR) method. The dihydropyridine calcium channel blocker nifedipine is classified as a poorly soluble drug; its most stable crystalline form is known as form I. Transmission spectra, collected from 0.2 to 2.0 THz (6.6 to 66 cm(-1) ), of nifedipine crystals had a strong absorption peak at 1.2 THz (40 cm(-1) ) at room temperature. When the nifedipine crystals were mixed with poloxamer 188 and suspended in water, the resulting spectra measured using the ATR method had a peak at the same frequency as in the spectra obtained in transmission mode. Furthermore, the peak area was proportional to the amount of crystals. The upward sloping baseline in the spectra, corresponding to water absorption, decreased stepwise with increasing amounts of crystalline particles. We confirmed that the spectra gave excellent quantitative results, using partial least-squares regression analysis. The results suggest the possibility of using this method for qualitative and quantitative assessments of crystalline drugs in suspension.

Terahertz time domain attenuated total reflection spectroscopy with an integrated prism system.

We demonstrated attenuated total reflection (ATR) spectroscopy with an integrated prism system that included a terahertz emitter, a terahertz receiver, and an ATR prism. The ATR prism had two internal off-axis parabolic mirrors for, respectively, collimating and focusing the terahertz waves. The Fresnel loss at each interface was reduced, and the total propagation efficiency was 3.36 times larger than when using a non-integrated prism system. The refractive index of water samples calculated from the experimental data showed good agreement with values reported in the literature.

Fast frequency-resolved terahertz imaging.

We propose a fast, frequency-resolved, real-time, terahertz imaging method. With our method, images at two specific terahertz frequencies can be acquired in 1 min. Three kinds of drugs (L-histidine, maltose, and CBZ3), which have absorption peaks in the terahertz region, were distinguished in 3 min by using our method. This technique can be used in industrial applications, such as nondestructive testing.