Embedding a Metal-Binding Motif for Copper Transporter into a Lipid Bilayer by Cu(I) Binding

Peptide–lipid interactions are widely involved with biologically significant phenomena, including the pathogenic mechanisms of protein misfolding diseases and transmembrane protein folding. In this paper, the interaction of the cysteine/tryptophan (Cys/Trp) motif, which is a metal-binding motif of copper transporter (Ctr) proteins, with a lipid bilayer was studied using fluorescence and circular dichroism (CD) spectroscopy. The binding of Cu­(I) to the Cys/Trp motif induced a large red-edge excitation shift in the Trp fluorescence, indicating that the Trp residue is located inside the lipid bilayer following complexation of Cu­(I) with the Cys/Trp motif. The Stern–Volmer quenching of the Trp fluorescence also supported the Cu­(I) binding peptide embedding in the lipid bilayer. The measurement of the CD spectra indicated the increase in β-sheet content of the Cys/Trp motif peptide as a result of Cu­(I) binding. These results lead to the conclusion that complexation with Cu­(I) induces the change in the secondary structure of the Cys/Trp motif, which results in the peptide embedding in the lipid bilayer. Cu­(I)-induced enhancement of the lipid affinity is discussed in terms of the mechanism for copper transport by Ctr

Experimental Evaluation of the Density of Water in a Cell by Raman Microscopy

We report direct observation of a spatial distribution of water molecules inside of a living cell using Raman images of the O–H stretching band of water. The O–H Raman intensity of the nucleus was higher than that of the cytoplasm, indicating that the water density is higher in the nucleus than that in the cytoplasm. The shape of the O–H stretching band of the nucleus differed from that of the cytoplasm but was similar to that of the balanced salt solution surrounding cells, indicating less crowded environments in the nucleus. The concentration of biomolecules having C–H bonds was also estimated to be lower in the nucleus than that in the cytoplasm. These results indicate that the nucleus is less crowded with biomolecules than the cytoplasm

Effects of Nanosecond Pulsed Electric Fields on the Intracellular Function of HeLa Cells As Revealed by NADH Autofluorescence Microscopy

The fluorescence lifetime of the endogenous fluorophore of reduced nicotinamide adenine dinucleotide (NADH) in HeLa cells is affected by the application of nanosecond pulsed electric fields (nsPEFs). In this study, we found that after nsPEF application, the fluorescence lifetime became longer and then decreased in a stepwise manner upon further application, irrespective of the pulse width in the range of 10–50 ns. This application time dependence of the NADH fluorescence lifetime is very similar to the time-lapse dependence of the NADH fluorescence lifetime following the addition of an apoptosis inducer, staurosporine. These results, as well as the membrane swelling and blebbing after the application of nsPEFs, indicate that apoptosis is also induced by the application of nsPEFs in HeLa cells. In contrast to the lifetime, the fluorescence intensity remarkably depended on the pulse width of the applied nsPEF. When the pulse width was as large as 50 ns, the intensity monotonically increased and was distributed over the entire cell as the application duration became longer. As the pulse width of the applied electric field became smaller, the magnitude of the field-induced increase in NADH fluorescence intensity decreased; the intensity was reduced by the electric field when the pulse width was as small as 10 ns. These results suggest that the mechanism of electric-field-induced apoptosis depends on the pulse width of the applied nsPEF

External Electric Field Effects on Excited-State Intramolecular Proton Transfer in 4′‑<i>N</i>,<i>N</i>‑Dimethylamino-3-hydroxyflavone in Poly(methyl methacrylate) Films

The external electric field effects on the steady-state electronic spectra and excited-state dynamics were investigated for 4′-<i>N</i>,<i>N</i>-(dimethylamino)-3-hydroxyflavone (DMHF) in a poly­(methyl methacrylate) (PMMA) film. In the steady-state spectrum, dual emission was observed from the excited states of the normal (N*) and tautomer (T*) forms. Application of an external electric field of 1.0 MV·cm^–1 enhanced the N* emission and reduced the T* emission, indicating that the external electric field suppressed the excited-state intramolecular proton transfer (ESIPT). The fluorescence decay profiles were measured for the N* and T* forms. The change in the emission intensity ratio N*/T* induced by the external electric field is dominated by ESIPT from the Franck–Condon excited state of the N* form and vibrational cooling in potential wells of the N* and T* forms occurring within tens of picoseconds. Three manifolds of fluorescent states were identified for both the N* and T* forms. The excited-state dynamics of DMHF in PMMA films has been found to be very different from that in solution due to intermolecular interactions in a rigid environment

pH-Dependent Network Formation of Quantum Dots and Fluorescent Quenching by Au Nanoparticle Embedding

A simple approach to the creation of colloidal assemblies is in high demand for the development of functional devices. Here, we present the preparation of CdTe-QD (quantum dot) networks in as little as 1 day simply by pH modification without the use of oxidants. The QD network was tractable in water and casting from a droplet produced a porous networked film on both hydrophobic and hydrophilic solid substrates. Further, we found that citrate-protected gold nanoparticles (AuNPs, <i>d</i> = 5 nm) could be incorporated into the QD networks to afford a QD/Au composite network, and that the fluorescence from the QDs was largely decreased by the addition of a small proportion of AuNPs (QD:AuNP = 99.4:0.6), probably due to the efficient charge transfer through the network. These data indicate that our method is suitable for application to the creation of metal/QD hybrid materials that can be integrated into wet-based processes