Construction of a Ca²⁺-Gated Artificial Channel by Fusing Alamethicin with a Calmodulin-Derived Extramembrane Segment

Using native chemical ligation, we constructed a Ca²⁺-gated fusion channel protein consisting of alamethicin and the C-terminal domain of calmodulin. At pH 5.4 and in the absence of Ca²⁺, this fusion protein yielded a burst-like channel current with no discrete channel conductance levels. However, Ca²⁺ significantly lengthened the specific channel open state and increased the mean channel current, while Mg²⁺ produced no significant changes in the channel current. On the basis of 8-anilinonaphthalene-1-sulfonic acid (ANS) fluorescent measurement, Ca²⁺-stimulated gating may be related to an increased surface hydrophobicity of the extramembrane segment of the fusion protein

<i>J</i>‑Resolved ¹H NMR 1D-Projections for Large-Scale Metabolic Phenotyping Studies: Application to Blood Plasma Analysis

¹H nuclear magnetic resonance (NMR) spectroscopy-based metabolic phenotyping is now widely used for large-scale epidemiological applications. To minimize signal overlap present in 1D ¹H NMR spectra, we have investigated the use of 2D <i>J</i>-resolved (JRES) ¹H NMR spectroscopy for large-scale phenotyping studies. In particular, we have evaluated the use of the 1D projections of the 2D JRES spectra (pJRES), which provide single peaks for each of the <i>J</i>-coupled multiplets, using 705 human plasma samples from the FGENTCARD cohort. On the basis of the assessment of several objective analytical criteria (spectral dispersion, attenuation of macromolecular signals, cross-spectral correlation with GC-MS metabolites, analytical reproducibility and biomarker discovery potential), we concluded that the pJRES approach exhibits suitable properties for implementation in large-scale molecular epidemiology workflows