ROS-Responsive Methionine-Containing Amphiphilic Peptides Impart Enzyme-Triggered Phase Transition and Antioxidant Cell Protection.

Reactive oxygen species (ROS) are produced by cellular activities, such as metabolism and immune response, and play important roles in cell signaling and homeostasis. However, overproduced ROS causes irreversible damage to nucleic acids and membrane lipids, supporting genetic mutations and enhancing the effects of aging. Cells defend themselves against ROS using antioxidant systems based on redox-active sulfur and transition metals. Inspired by such biological redox-responsive systems, we developed methionine-containing self-assembling peptides. The Met-containing peptides formed hydrogels that underwent a gel-to-sol phase transition upon oxidation by H2O2, and the sensitivity of the peptides to the oxidant increased as the number of Met residues increased. The peptide containing three Met residues, the largest number of Met residues in our series of designed peptides, showed the highest sensitivity to oxidation and detoxification to protect cells from ROS damage. In addition, this peptide underwent a phase transition in response to H2O2 produced by an oxidizing enzyme. This study demonstrates the design of a supramolecular biomaterial that is responsive to enzymatically generated ROS and can protect cells against oxidative stress.

Origin of unique hyper-Raman signals of trifluoroethanol.

We report the hyper-Raman (HR) spectrum of trifluoroethanol, excited with 532 nm light, in an aqueous solution at basic pH. The HR spectrum exhibits a distinct spectral pattern that diverges entirely from the infrared and Raman spectra of trifluoroethanol. This observed unique HR signal was attributed to the products of photoinduced radical reactions in the aqueous solution. This result exemplifies the exceptional capabilities of HR spectroscopy based on resonance conditions.

Determination of sugar content in honey using LC-Raman and programmable pump-Raman methods.

We combined (i) liquid chromatography and Raman spectrometry (LC-Raman) and (ii) programmable pump and Raman spectrometry (PP-Raman) to separate and identify compounds in a mixture. These techniques were applied to conduct a quantitative analysis of the sugars in honey. The spectral and temporal axes of the LC-Raman data were analyzed using the MCR-ALS analysis procedure, which enabled the separation and identification of four sugars (glucose, fructose, sucrose, and trehalose). The PP-Raman method was employed to examine the sugar concentration dependence of the intensity pattern of the Raman spectrum, and the linear concentration dependence of the intensity was obtained. The sugar contents were quantitatively determined from the integrated area of the elution peaks. The result was consistent with those derived from mass spectrometry and previous studies. The origin of the errors in the derived sugar contents is discussed. Our study presents a novel quantitative LC-Raman spectrometric method that does not rely on resonance or surface enhancement effects.

ROS-Triggered Gel-Sol Transition and Kinetics-Controlled Cargo Release by Methionine-Containing Peptides.

The gel-sol transition of self-assembling peptides is a useful switch for environment-dependent drug release. For their applications, kinetics control of the responses is important for matching the velocity of release to the target biological events. Here we demonstrate the chemical control of redox-triggered gel-sol transition kinetics of self-assembling peptides by altering the amino acid sequence. Amphiphilic peptides were developed in which a methionine residue was located in the middle (JigSAP-IMI) or near the N terminus (JigSAP-MII). Both peptides formed hydrogels under physiological conditions-forming ß-sheet-based supramolecular nanofibers. In contrast, the oxidized forms remained in the solution state under identical conditions-adopting α-helix-rich secondary structures. Upon oxidation with H2 O2 , a reactive oxygen species, JigSAP-MII showed a faster gel-to-sol transition and cargo-releasing than JigSAP-IMI, thus indicating that the phase-transition and releasing kinetics of self-assembling peptides can be rationally controlled by the position of the reactive amino acid residue.

Resonance Hyper-Raman Spectroscopy of Nucleotides and Polynucleotides.

We applied 532 nm-excited two-photon resonance hyper-Raman (RHR) spectroscopy to nucleotides (dA, dG, dT, and dC) to obtain fundamental knowledge about their spectral patterns. The RHR spectrum of each nucleotide exhibited various modes of the purine and pyrimidine rings, showing the ability to acquire the structural information on the chromophore. The band positions of the RHR spectrum and the 266 nm-excited one-photon UV-resonance Raman (UVRR) spectrum were identical, while the intensity patterns differed. The peak assignments of the RHR bands were given by analogy to the UVRR spectrum. In examining the polynucleotides, which form a double-stranded helix through intermolecular hydrogen bonds, some RHR bands were found to be available as structural markers. Moreover, several overtone and combination bands were detected above 2000 cm-1. The frequencies of dA and dG were accounted for by considering the involvement of the vibration of dA at 1579 cm-1 and that of dG at 1482 cm-1, respectively. Multiple vibronically active modes were seen for dT and dC. HR spectroscopy offers unique information on the fundamental, combination, and overtone modes of dA and dG, of which the multiple electronic states are involved in the resonance process.

Efficient protein incorporation and release by a jigsaw-shaped self-assembling peptide hydrogel for injured brain regeneration.

During injured tissue regeneration, the extracellular matrix plays a key role in controlling and coordinating various cellular events by binding and releasing secreted proteins in addition to promoting cell adhesion. Herein, we develop a cell-adhesive fiber-forming peptide that mimics the jigsaw-shaped hydrophobic surface in the dovetail-packing motif of glycophorin A as an artificial extracellular matrix for regenerative therapy. We show that the jigsaw-shaped self-assembling peptide forms several-micrometer-long supramolecular nanofibers through a helix-to-strand transition to afford a hydrogel under physiological conditions and disperses homogeneously in the hydrogel. The molecular- and macro-scale supramolecular properties of the jigsaw-shaped self-assembling peptide hydrogel allow efficient incorporation and sustained release of vascular endothelial growth factor, and demonstrate cell transplantation-free regenerative therapeutic effects in a subacute-chronic phase mouse stroke model. This research highlights a therapeutic strategy for injured tissue regeneration using the jigsaw-shaped self-assembling peptide supramolecular hydrogel.

Regulation of Cell Volume by Nanosecond Pulsed Electric Fields.

Stimulation of cells by nanosecond pulsed electric fields (nsPEFs) has attracted attention as a technology for medical applications such as cancer treatment. nsPEFs have been shown to affect intracellular environments without significant damage to cell membranes; however, the mechanism underlying the effect of nsPEFs on cells remains unclear. In this study, we constructed electrodes for applying nsPEFs and analyzed the change in volume of a single cell due to nsPEFs using fluorescence and Raman microscopy. It was shown that the direction of the change depended on the applied electric field; expansion due to the influx of water was observed at high electric field, and cell shrinkage was observed at low electric field. The change in cell volume was correlated to the change in the intracellular Ca2+ concentration, and nsPEFs-induced shrinking was not observed when the Ca2+-free medium was used. This result suggests that the cell shrinkage is related to the regulatory volume decrease where the cell adjusts the increase in intracellular Ca2+ concentration, inducing the efflux of ions and water from the cell.

Hydrogel-Stiffening and Non-Cell Adhesive Properties of Amphiphilic Peptides with Central Alkylene Chains.

Invited for the cover of this issue is the group of Takahiro Muraoka at Tokyo University of Agriculture and Technology and collaborators. The image depicts nanofiber formation of an amphiphilic peptide with a central alkylene chain that shows non-cell adhesive properties. Read the full text of the article at 10.1002/chem.202100739.

Hydrogel-Stiffening and Non-Cell Adhesive Properties of Amphiphilic Peptides with Central Alkylene Chains.

Amphiphilic peptides bearing terminal alkyl tails form supramolecular nanofibers that are increasingly used as biomaterials with multiple functionalities. Insertion of alkylene chains in peptides can be designed as another type of amphiphilic peptide, yet the influence of the internal alkylene chains on self-assembly and biological properties remains poorly defined. Unlike the terminal alkyl tails, the internal alkylene chains can affect not only the hydrophobicity but also the flexibility and packing of the peptides. Herein, we demonstrate the supramolecular and biological effects of the central alkylene chain length inserted in a peptide. Insertion of the alkylene chain at the center of the peptide allowed for strengthened ß-sheet hydrogen bonds and modulation of the packing order, and consequently the amphiphilic peptide bearing C2 alkylene chain formed a hydrogel with the highest stiffness. Interestingly, the amphiphilic peptides bearing internal alkylene chains longer than C2 showed a diminished cell-adhesive property. This study offers a novel molecular design to tune mechanical and biological properties of peptide materials.

pH-controlled stacking direction of the ß-strands in peptide fibrils.

Peptides provide a framework for generating functional biopolymers. In this study, the pH-dependent structural changes in the 21-29 fragment peptide of ß2-microglobulin (ß2m21-29) during self-aggregation, i.e., the formation of an amyloid fibril, were discussed. The ß-sheet structures formed during parallel stacking under basic conditions (pH ≥ 7.7) adopted an anti-parallel stacking configuration under acidic conditions (pH ≤ 7.6). The parallel and anti-parallel ß-sheets existed separately at the intermediate pH (pH = 7.6-7.7). These results were attributed to the rigidity of the ß-sheets in the fibrils, which prevented the stable hydrogen bonding interactions between the parallel and anti-parallel ß-sheet moieties. This observed pH dependence was ascribed to two phenomena: (i) the pH-dependent collapse of the ß2m21-29 fibrils, which consisted of 16 ± 3 anti-parallel ß-sheets containing a total of 2000 ß-strands during the deprotonation of the NH3+ group (pKa = 8.0) of the ß-strands that occurred within 0.7 ± 0.2 strands of each other and (ii) the subsequent formation of the parallel ß-sheets. We propose a framework for a functional biopolymer that could alternate between the two ß-sheet structures in response to pH changes.

Online Liquid Chromatography-Raman Spectroscopy Using the Vertical Flow Method.

Liquid chromatography and Raman spectroscopy (LC-Raman system) were combined and developed with the aid of the vertical flow method that enhances the Raman signal intensity. The LC-Raman system enabled the online acquisition of the nonresonance Raman spectrum of LC eluates. We employed singular value decomposition (SVD) and subsequent reconstruction of the components for the analysis of two-dimensional (temporal and spectral) data. The obtained components were consistent with the Raman spectra and elution patterns of the samples, indicating the appropriateness of the SVD-based procedure. The rise and fall times of the elution band of the temporal component were considered as the instrumental function. D2O mixed with H2O exhibited increased full width at half maximum of the elution band of up to 30% in comparison to the calculated value because of diffusion. Band broadening was less significant in the case in which an immiscible solute (pentane) was mixed with H2O. The limits of detection and quantitation were 1.2 ± 0.1, 2.1 ± 0.1, and 2.7 ± 0.1 mM and 4.1 ± 0.1, 6.9 ± 0.1, and 9.1 ± 0.2 mM for the ortho-, meta-, and para-isomers of methoxyphenol, respectively. The nonresonance Raman experiment provides the molecular specificity to LC on the basis of the inherent properties of eluates.

Linear, mixed-valent homocatenated tri-tin complexes featuring Sn-Sn bonds.

A series of tri-tin complexes (LPhSn)3X with triple-decker structures (LPh = 2,5-di(o-pyridyl)-3,4-diphenylpyrrolate; X = Cl, AlCl4, OTf, and PF6) was synthesized by reducing LPhSnCl with LiBsBu3H and subsequent reactions. Structural characterization of (LPhSn)3Cl revealed a Sn-Sn-Sn core, and DFT calculations suggest that its HOMO is primarily σ-bonding along the tri-tin framework. (LPhSn)3Cl reacts with W(CO)5THF to afford (LPhSn)2(W(CO)5)2 and LPhSnCl, implying that (LPhSn)3Cl may exhibit dynamic behavior in solution.

Time-resolved FTIR study on the structural switching of human galectin-1 by light-induced disulfide bond formation.

Disulfide bonds play a fundamental role in controlling the tertiary structure of proteins; the formation or cleavage of some disulfide bonds can switch the structures and/or functions of proteins. Human galectin-1 (hGal-1), which is a lectin protein, exemplifies how both structure and function are changed by disulfide bonds; the structure and sugar-binding ability of hGal-1 are altered by the formation and cleavage of its three intra-molecular disulfide bonds. In the present study, the dynamics of the structural change of hGal-1 by the formation of disulfide bonds were investigated by time-resolved FTIR spectroscopy combined with a modification in which its thiol groups (-SH) were replaced with S-nitrosylated groups (SNO). Photodissociation of NO from SNO in reduced hGal-1 induced disulfide bond formation and transformed it into the oxidised form. The structural change to the oxidised form involved three distinct kinetics with fast (<300 s), middle (∼600 s), and slow (∼6400 s) lifetimes. In an examination of hGal-1 in the lactose-bound form, structural changes owing to the release of substrate lactose were also observed upon disulfide bond formation. The present method using the photodissociation of NO is useful for monitoring the dynamics of structural changes following disulfide formation.

Tautomer Structures in Ketose-Aldose Transformation of 1,3-Dihydroxyacetone Studied by Infrared Electroabsorption Spectroscopy.

The acyclic form of monosaccharides exists in a structural equilibrium, with aldose having the aldehyde group and ketose the ketone group (ketose-aldose equilibrium). A basic catalyst facilitates their transformation, which affects the chemical properties of the monosaccharide. In this study, we investigated the ketose-aldose transformation of 1,3-dihydroxyacetone (1,3-DHA), one of the simplest systems of the ketose-aldose equilibrium. We examined the effects of piperidine as the basic catalyst and used IR electroabsorption spectroscopy to study the responses to an external electric field. We analyzed the changes in IR absorption by considering the changes in the molecular orientation and number of molecules in response to the external electric field. The results of the analysis revealed the permanent dipole moment µP, an angle η between µP and µT (the transition moment of the molecular vibration), and the equilibrium constants. The ketose-aldose transformation of 1,3-DHA can be explained in terms of the equilibrium of three states. In the presence of piperidine, a five-state equilibrium was concluded. On the basis of the experimental data, we propose plausible models of dihydroxyacetone, E-enediols, Z-enediol, or glyceraldehyde for each state. The results of our structural analysis of these tautomers provide a detailed understanding of the ketose-aldose transformation of acyclic saccharides and the effects of the basic catalyst.

A Vertical Flow Method for Sensitive Raman Protein Measurement in Aqueous Solutions.

Raman scattering is intrinsically faint. Raman spectroscopy would be more valuable with improvements in the signal detection efficiency. To improve the signal detection efficiency, we propose a vertical flow method, which is a derivative of the liquid core optical fiber technique employed for sensitive Raman signal detection. In the vertical flow method, the sample solution flows from a pinhole to prepare a liquid column wrapped with air and uses total reflection at the sample-air interface to confine the excitation beam and Raman signal. The Raman signal emerges from the pinhole for efficient collection. This method enhanced Raman signal intensity by up to 90 times. When measuring a bovine serum albumin aqueous solution, the limit of detection (LOD) was 0.029 ± 0.003 mg mL-1 (0.44 ± 0.05 µM). We discuss the signal enhancement factor dependence on several parameters in the vertical flow method. Our method provides a simple technique to improve Raman spectroscopy sensitivity using universal materials.

Preparation and photo-induced activities of water-soluble amyloid ß-C60 complexes.

We have shown that fullerene (C60) becomes soluble in water by mixing fullerene and amyloid ß peptide (Aß40) whose fibril structures are considered to be associated with Alzheimer's disease. The water-solubility of fullerene arises from the generation of a nanosized complex between fullerene and the monomer species of Aß40 (Aß40-C60). The prepared Aß40-C60 exhibits photo-induced activity with visible light to induce the inhibition of Aß40 fibrillation and the cytotoxicity for cultured HeLa cells. The observed photo-induced phenomena result from the generation of singlet oxygen via photoexcitation, inducing oxidative damage to Aß40 and HeLa cells. The oxidized Aß40 following photoexcitation of Aß40-C60 was confirmed by mass spectrometry.

Directly Probing Intermolecular Structural Change of a Core Fragment of ß2-Microglobulin Amyloid Fibrils with Low-Frequency Raman Spectroscopy.

Amyloid fibrils, which are ordered aggregates of proteins or peptides, have attracted keen interest because their deposition causes serious human diseases. Despite many studies utilizing X-ray crystallography, solid-state NMR, and other methods, intermolecular interactions governing the fibril formation remain largely unclear. Here, we used low-frequency Raman (LFR) spectroscopy to investigate the intermolecular ß-sheet structure of a core fragment of ß2-microglobulin amyloid fibrils, ß2m21-29, in aqueous buffer solutions. The LFR spectra (approximately 10-200 cm-1) of ß2m21-29 amyloid fibrils measured at different pH values (ranging from 6.8 to 8.0) revealed a broad-spectral pattern with a maximum at ∼80 cm-1 below pH 7.2 and at ∼110 cm-1 above pH 7.4. This observation is attributed to a pH-dependent structural change from an antiparallel to a parallel intermolecular ß-sheet structure. Multivariate curve resolution-alternating least-squares (MCR-ALS) analysis enabled us to decompose the apparently monotonous LFR spectra into three distinctly different contributions: intermolecular vibrations of the parallel and antiparallel ß-sheets and intramolecular vibrations of the peptide backbone. Peak positions of the obtained LFR bands not only exhibit a much more pronounced difference between the two ß-sheets than the conventional amide I band, but they also suggest stronger intermolecular interaction, due presumably to the hydrophobic effect, in the parallel ß-sheet than in the antiparallel ß-sheet. The present results show that LFR spectroscopy in combination with the MCR-ALS analysis holds promise for real-time tracking of the intermolecular dynamics of amyloid fibril formation under physiological conditions.

Citrullination and deamidation affect aggregation properties of amyloid ß-proteins.

Citrullination and deamidation, which are aging-related posttranslational modifications, increase the number of negative charges on amyloid ß-protein (Aß) at neutral pH. We investigated the effects of these modifications on the fibrillation properties of Aß. The Arg5→Cit modification of Aß1-40 did not affect the fibrillation rate, and brought ß-sheet structures unlike that in the Aß1-40 fibril. The Asn27→Asp modification of Aß1-40 stopped the fibrillation and induced the formation of aggregates that involved an anti-parallel ß-sheet. Aß1-42 with the Arg5→Cit modification showed increased solubility in aqueous media, and its fibril formation became slower than that of Aß1-42. The modification did not change the parallel ß-sheet structure of the fibrils. Aß1-42 with the Asn27→Asp modification partially formed fibrils that involved the parallel ß-sheet structure. Using the thioflavin T (ThT) assay, an increased fraction of the soluble oligomer of each Aß analog was transiently detected during fibrillation. An increase in the number of negative charges at basic pH affected the aggregation properties of Aß in a manner different from that with the modifications, suggesting that change in properties of the posttanslationally modified residues rather than the number of charges in the peptide was important.

Generation of self-clusters of galectin-1 in the farnesyl-bound form.

Ras protein is involved in a signal transduction cascade in cell growth, and cluster formation of H-Ras and human galectin-1 (Gal-1) complex is considered to be crucial to achieve its physiological roles. It is considered that the complex is formed through interactions between Gal-1 and the farnesyl group (farnesyl-dependent model), post-translationally modified to the C-terminal Cys, of H-Ras. We investigated the role of farnesyl-bound Gal-1 in the cluster formation by analyzing the structure and properties of Gal-1 bound to farnesyl thiosalicylic acid (FTS), a competitive inhibitor of the binding of H-Ras to Gal-1. Gal-1 exhibited self-cluster formation upon interaction with FTS, and small- and large-size clusters were formed depending on FTS concentration. The galactoside-binding pocket of Gal-1 in the FTS-bound form was found to play an important role in small-size cluster formation. Large-size clusters were likely formed by the interaction among the hydrophobic sites of Gal-1 in the FTS-bound form. The present results indicate that Gal-1 in the FTS-bound form has the ability to form self-clusters as well as intrinsic lectin activity. Relevance of the self-clustering of FTS-bound Gal-1 to the cluster formation of the H-Ras-Gal-1complex was discussed by taking account of the farnesyl-dependent model and another (Raf-dependent) model.

Effects of N-Methylated Amyloid-ß30-40 Peptides on the Fibrillation of Amyloid-ß1-40.

Alzheimer's disease is a neurodegenerative disorder associated with amyloid-ß (Aß) fibrillation. N-Methylated amyloid-ß peptides are potent inhibitors of amyloid-ß fibrillation. We investigated the inhibitory effect of N-Methylated Aß30-40 peptides on Aß1-40 fibrillation. N-Methylated Aß30-40 peptides affected the fibrillation, and this effect was dependent on the concentration of N-Methylated peptide and the number and position of N-Methylated groups. N-Methylated Aß30-40 peptides were co-aggregated with Aß1-40 . Spectroscopic technique was adopted to investigate an origin of the observed dependence. Suppression of thioflavin T (ThT) fluorescence count was correlated with the dissociation constant Kd of monomer-dimer equilibrium of each N-Methylated Aß30-40 peptide. Monomeric N-Methylated peptides decreased ThT fluorescence count during Aß1-40 fibrillation. Secondary structure content was not largely different between Aß1-40 fibrils and co-aggregates. These results suggested that N-Methylated Aß30-40 peptides disrupted the regular ß-sheet structure of Aß1-40 fibrils and affected the ThT fluorescence count. The monomer-dimer equilibrium of N-Methylated peptides was (partly) responsible for the observed dependence of their inhibitory effect on the concentration of N-Methylated peptide and the number and position of N-Methylated groups. Our study provides a hint to design new N-Methylated inhibitor peptides of fibrillation.