Multidimensional vibrational circular dichroism for insect wings: Comparison of species.

This study reports the microscopic measurements of vibrational circular dichroism (VCD) on four different insect wings using a quantum cascade laser VCD system equipped with microscopic scanning capabilities (named multi-dimensional VCD [MultiD-VCD]). Wing samples, including (i) beetle, Anomala albopilosa (female), (ii) European hornet, Verspa crabro flavofasciata Cameron, 1903 (female), (iii) tiny dragonfly, Nannophya pygmae Rambur, 1842 (male), and (iv) dragonfly, Symetrum gracile Oguma, 1915 (male), were used in this study. Two-dimensional patterns of VCD signals (~10 mm × 10 mm) were obtained at a spatial resolution of 100 µm. Measurements covered the absorption peaks assigned to amides I and II in the range of 1500-1740 cm-1 . The measurements were based on the enhancement of VCD signals for the stereoregular linkage of peptide groups. The patterns were remarkably dependent on the species. In samples (i) and (ii), the wings comprised segregated domains of protein aggregates of different secondary structures. The size of each microdomain was approximately 100 µm. In contrast, no clear VCD spectra were detected in samples (iii) and (iv). One possible reason was that the chain of stereoregular polypeptides was too short to achieve VCD enhancement in samples (iii) and (iv). Notably, the unique features were only observed in the VCD spectra because the IR spectra were nearly the same among the species. The VCD results hinted at the connection of protein microscopic structures with the wing flapping mechanisms of each species.

Synthetic Nanocelluloses Fluorescently Responsible to Enzymatic Degradation.

Crystalline assemblies of cellulose and cellulose derivatives that can be synthetically produced by in vitro enzymatic reactions in a bottom-up manner have attracted increasing attention as chemically designable functional nanomaterials (e.g., synthetic nanocelluloses). In this study, we demonstrate the preparation and characterization of alkyl ß-celluloside assemblies loaded with fluorescent molecules, which are fluorescently responsible to the enzymatic degradation of the cellulose moieties. The fluorescent properties are afforded to the assemblies by their bilayer-structured nanosheet morphologies realized through the uptake of environmentally responsive fluorescent molecules (namely, Nile Red (NR)). Incubation of the NR-loaded n-octyl ß-celluloside (CEL-C8) assembly with cellulase resulted in decreases in the fluorescence intensities. This suggests that NR molecules were released into the aqueous phase through enzymatic degradation of the cellulose moieties of CEL-C8 molecules in the assembly. The fluorescence decrease rates were clearly dependent on the concentration and source of cellulase. Fluorescence decreases through enzymatic degradation were again observed in the presence of contaminant proteins. These observations revealed the high potential of alkyl ß-celluloside assemblies loaded with fluorescent molecules as fluorescently responsible cellulase substrates for cellulase detection assays by simply measuring changes in the fluorescence intensities. Moreover, the assemblies were revealed as carriers for the controlled release of loaded molecules triggered by enzymatic degradation.

Dynamic membrane interaction and amyloid fibril formation of glucagon, melittin and human calcitonin.

Glucagon is a 29-amino acid peptide hormone secreted by pancreatic α-cells and interacts with specific receptors located in various organs. Glucagon tends to form gel-like fibril aggregates that are cytotoxic. It is important to reveal the glucagon-membrane interaction to understand activity and cytotoxicity of glucagon and glucagon oligomers. In this review, first glucagon-membrane interactions are described as morphological changes in dimyristoylphosphatidylcholine (DMPC) bilayers containing glucagon in acidic and neutral conditions as compared to the case of melittin. Second, fibril formation by glucagon in acidic solution is discussed in light of morphological and structural changes. Third, kinetic analysis of glucagon fibril formation was performed using a two-step autocatalytic reaction mechanism, as investigated in the case of human calcitonin. The first step is a nuclear formation, and the second step is an autocatalytic fibril elongation. Forth, fibril formation of glucagon inside glucagon-DMPC bilayers in neutral solution under near physiological condition is described.

Gelatin acrylamide with improved UV crosslinking and mechanical properties for 3D biofabrication.

Photocrosslinkable gelatin has attracted increasing interest in the field of biofabrication, with the most studied and widely used photocrosslinkable gelatin being gelatin methacrylate (GelMa). However, the 3D fabrication of GelMa has presented several limitations and challenges, primarily due to its slow crosslinking speed. It is generally known that acryl-based functional groups have faster reaction kinetics than methacryl-base groups. However, gelatin acrylamide (GelAc) has not been widely investigated, largely due to its increased complexity of synthesis relative to GelMA. In this study, we developed a novel synthesis method for GelAc. By varying the reaction ratio of reagents, GelAc with a degree of substitution from 20% to 95% was produced. The UV crosslinking properties of GelAc was studied, demonstrating significantly faster crosslinking kinetics than GelMa, especially at lower concentrations and low photoinitiator concentrations. The swelling ratio and mechanical properties of the crosslinked GelAc hydrogel were also characterized, and biocompatibility experiments conducted via both surface seeding and hydrogel encapsulation of cells, with good cell viability observed. The application of GelAc for 3D biofabrication was demonstrated by 3D printing. GelAc can be a useful material for the fabrication of 3D conduits for tissue engineering applications.

Evaluating the Stability of Cellulose Nanofiber Pickering Emulsions Using MRI and Relaxometry.

Magnetic resonance imaging (MRI) relaxometry and diffusion methods were used to highlight the instability mechanisms of oil-in-water Pickering emulsions stabilized by cellulose nanofibers (CNFs). Four different Pickering emulsions using different oils (n-dodecane and olive oil) and concentrations of CNFs (0.5 and 1.0 wt %) were systematically investigated over a period of one month after emulsification. The separation into a free oil, emulsion layer, and serum layer and the distribution of flocculated/coalesced oil droplets in several hundred micrometers were captured in MR images using fast low-angle shot (FLASH) and rapid acquisition with relaxation enhancement (RARE) sequences. The components of the Pickering emulsions (e.g., free oil, emulsion layer, oil droplets, and serum layer) were observable by different voxelwise relaxation times and apparent diffusion coefficients (ADCs) and reconstructing in the apparent T1, T2, and ADC maps. The mean T1, T2, and ADC of the free oil and serum layer corresponded well with MRI results for pure oils and water, respectively. Comparing the relaxation properties and translational diffusion coefficients of pure dodecane and olive oil obtained from NMR and MRI resulted in similar T1 and ADC but significantly different T2 depending on the sequence used. The diffusion coefficients of olive oil measured by NMR were much slower than dodecane. The ADC of the emulsion layer for dodecane emulsions did not correlate with the viscosity of the emulsions as the CNF concentration increased, suggesting the effects of restricted diffusion of oil/water molecules due to droplet packing.

Characterization of retinal chromophore and protonated Schiff base in Thermoplasmatales archaeon heliorhodopsin using solid-state NMR spectroscopy.

Heliorhodopsin (HeR) is a seven-helical transmembrane protein with a retinal chromophore that corresponds to a new rhodopsin family. HeR from the archaebacterium Thermoplasmatales archaeon (TaHeR) exhibits unique features, such as the inverted protein orientation in the membrane compared to other rhodopsins and a long photocycle. Here, we used solid-state nuclear magnetic resonance (NMR) spectroscopy to investigate the 13C and 15N NMR signals of the retinal chromophore and protonated Schiff base (RPSB) in TaHeR embedded in POPE/POPG membrane. Although the 14- and 20-13C retinal signals indicated 13-trans/15-anti (all-trans) configurations, the 20-13C chemical shift value was different from that of other microbial rhodopsins, indicating weakly steric hinderance between Phe203 and the C20 methyl group. 15N RPSB/λmax plot deviated from the linear correlation based on retinylidene-halide model compounds. Furthermore, 15N chemical shift anisotropy (CSA) suggested that Ser112 and Ser234 polar residues distinguish the electronic environment tendencies of RPSB from those of other microbial rhodopsins. Our NMR results revealed that the retinal chromophore and the RPSB in TaHeR exhibit unique electronic environments.

Cell-Free Expression of De Novo Designed Peptides That Form ß-Barrel Nanopores.

Nanopore sensing has attracted much attention as a rapid, simple, and label-free single-molecule detection technology. To apply nanopore sensing to extensive targets including polypeptides, nanopores are required to have a size and structure suitable for the target. We recently designed a de novo ß-barrel peptide nanopore (SVG28) that constructs a stable and monodispersely sized nanopore. To develop the sizes and functionality of peptide nanopores, systematic exploration is required. Here we attempt to use a cell-free synthesis system that can readily express peptides using transcription and translation. Hydrophilic variants of SVG28 were designed and expressed by the PURE system. The peptides form a monodispersely sized nanopore, with a diameter 1.1 or 1.5 nm smaller than that of SVG28. Such cell-free synthesizable peptide nanopores have the potential to enable the systematic custom design of nanopores and comprehensive sequence screening of nanopore-forming peptides.

Solid-state NMR for the characterization of retinal chromophore and Schiff base in TAT rhodopsin embedded in membranes under weakly acidic conditions.

TAT rhodopsin extracted from the marine bacterium SAR11 HIMB114 has a characteristic Thr-Ala-Thr motif and contains both protonated and deprotonated states of Schiff base at physiological pH conditions due to the low pKa. Here, using solid-state NMR spectroscopy, we investigated the 13C and 15N NMR signals of retinal in only the protonated state of TAT in the 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine/1-palmitoyl-2-oleoyl-sn-glycero-3-phospho (1'-rac-glycerol) (POPE/POPG) membrane at weakly acidic conditions. In the 13C NMR spectrum of 13C retinal-labeled TAT rhodopsin, the isolated 14-13C signals of 13-trans/15-anti and 13-cis/15-syn isomers were observed at a ratio of 7:3. 15N retinal protonated Schiff base (RPSB) had a significantly higher magnetic field resonance at 160 ppm. In 15N RPSB/λmax analysis, the plot of TAT largely deviated from the trend based on the retinylidene-halide model compounds and microbial rhodopsins. Our findings indicate that the RPSB of TAT forms a very weak interaction with the counterion.

Total Internal Reflection Raman Spectra of Alamethicin Interacting with Supported Lipid Bilayers at a Silica/Water Interface.

We report total internal reflection (TIR)-Raman spectroscopy to study intermolecular interactions between membrane-binding peptides and lipid bilayer membranes. The method was applied to alamethicin (ALM), a model peptide for channel proteins, interacting with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) bilayer membranes at a silica/water interface. After a dimethyl sulfoxide (DMSO) solution of ALM was added into the water subphase of the DPPC/DPPC bilayer, Raman signals in the CH stretching region increased in intensity reflecting the appearance of the Raman bands due to ALM and DMSO. To identify ALM-dependent spectral changes, we removed DPPC and DMSO contributions from the Raman spectra. We first subtracted the spectrum of the DPPC bilayer from those after the addition of the ALM solution. The contribution of DMSO was then removed by subtracting a DMSO spectrum from the resultant spectra. The DMSO spectrum was obtained in a similar way from a control experiment where DMSO alone was added into the subphase. With the use of this double difference approach, the ALM-dependent changes were successfully obtained. Experiments with DPPC bilayers with deuterated acyl chains revealed that most of the spectral change observed after the addition of ALM was due to the vibrational bands of ALM, not originated from ALM-induced conformational changes of the lipid bilayers.

Alkyl chain length-dependent protein nonadsorption and adsorption properties of crystalline alkyl ß-celluloside assemblies.

Cellulose-based crystalline assemblies artificially constructed in a bottom-up manner are attracting increasing attention as chemically stable and functionally designable nano- to macroscale materials. However, basic knowledge of how such crystalline assemblies interact with biomolecules and how to control them via molecular design is still limited. In this study, we investigated the protein adsorption properties of crystalline lamella assemblies composed of alkyl ß-cellulosides (namely, ethyl, n-butyl, and n-hexyl ß-cellulosides) or plain cellulose, which all have an antiparallel molecular arrangement. It was found that the adsorption of proteins was observed only for the n-hexyl ß-celluloside assembly, while it was hardly observed for other assemblies. The n-hexyl groups appeared to be ordinarily embedded in the assembly surface in an aqueous phase, while, when in contact with proteins, n-hexyl groups appeared to be tethered to promote protein adsorption. All-atom molecular dynamics simulations supported the contradictory protein adsorption properties. The basic knowledge obtained herein is highly valuable for controlling the interactions of cellulose-based synthetic assemblies with proteins for designing new biological applications.

Advanced Research on Structure-Function Relationships of Membrane Proteins.

Membrane proteins embedded in biological membranes account for 30% of the proteins encoded in the human genome and play an essential role in maintaining the homeostasis of cells by functioning as transporters, for signal transaction and energy conversion, amongst other functions [...].

Photoreaction Pathways of Bacteriorhodopsin and Its D96N Mutant as Revealed by in Situ Photoirradiation Solid-State NMR.

Bacteriorhodopsin (BR) functions as a light-driven proton pump that transitions between different states during the photocycle, such as all-trans (AT; BR568) and 13-cis, 15-syn (CS; BR548) state and K, L, M1, M2, N, and O intermediates. In this study, we used in situ photoirradiation 13C solid-state NMR to observe a variety of photo-intermediates and photoreaction pathways in [20-13C]retinal-WT-BR and its mutant [20-13C, 14-13C]retinal-D96N-BR. In WT-BR, the CS state converted to the CS* intermediate under photoirradiation with green light at -20 °C and consequently converted to the AT state in the dark. The AT state converted to the N intermediate under irradiation with green light. In D96N-BR, the CS state was converted to the CS* intermediate at -30 °C and consequently converted to the AT state. Simultaneously, the AT state converted to the M and L intermediates under green light illumination at -30 °C and subsequently converted to the AT state in the dark. The M intermediate was directly excited to the AT state by UV light illumination. We demonstrated that short-lived photo-intermediates could be observed in a stationary state using in situ photoirradiation solid-state NMR spectroscopy for WT-BR and D96N-BR, enabling insight into the light-driven proton pump activity of BR.

De novo design of a nanopore for single-molecule detection that incorporates a ß-hairpin peptide.

The amino-acid sequence of a protein encodes information on its three-dimensional structure and specific functionality. De novo design has emerged as a method to manipulate the primary structure for the development of artificial proteins and peptides with desired functionality. This paper describes the de novo design of a pore-forming peptide, named SV28, that has a ß-hairpin structure and assembles to form a stable nanopore in a bilayer lipid membrane. This large synthetic nanopore is an entirely artificial device for practical applications. The peptide forms multidispersely sized nanopore structures ranging from 1.7 to 6.3 nm in diameter and can detect DNAs. To form a monodispersely sized nanopore, we redesigned the SV28 by introducing a glycine-kink mutation. The resulting redesigned peptide forms a monodisperse pore with a diameter of 1.7 nm leading to detection of a single polypeptide chain. Such de novo design of a ß-hairpin peptide has the potential to create artificial nanopores, which can be size adjusted to a target molecule.

A hybrid strategy combining solution NMR spectroscopy and isothermal titration calorimetry to characterize protein-nanodisc interaction.

NMR is a powerful tool for characterizing intermolecular interactions at atomic resolution. However, the nature of the complex interactions of membrane-binding proteins makes it difficult to elucidate the interaction mechanisms. Here, we demonstrated that structural and thermodynamic analyses using solution NMR spectroscopy and isothermal titration calorimetry (ITC) can clearly detect a specific interaction between the pleckstrin homology (PH) domain of ceramide transport protein (CERT) and phosphatidylinositol 4-monophosphate (PI4P) embedded in the lipid nanodisc, and distinguish the specific interaction from nonspecific interactions with the bulk surface of the lipid nanodisc. This NMR-ITC hybrid strategy provides detailed characterization of protein-lipid membrane interactions.

Mapping of Supramolecular Chirality in Insect Wings by Microscopic Vibrational Circular Dichroism Spectroscopy: Heterogeneity in Protein Distribution.

The supramolecular chirality of the hindwing of Anomala albopilosa (male) was investigated using a microscopic vibrational circular dichroism (VCD) system, denoted as MultiD-VCD. The source of intense infrared (IR) light for the system was a quantum cascade laser. Two-dimensional maps of IR and VCD spectra were taken by scanning the surface area (ca. 2 mm × 2 mm) of the insect hindwing tissue. The spectra ranged from 1500 to 1700 cm-1, and the maps have a spatial resolution of 100 µm. The distribution of proteins, including their supramolecular structures, was analyzed from the location-dependent spectral shape of the VCD bands assigned to amides I and II. The results revealed that the hindwing consists of segregated domains of proteins with different secondary structures: an α-helix (in one part of the membrane), a hybrid of α-helix and ß-sheet (in another part of the membrane), and a coil (in a vein).

Structure of a retinal chromophore of dark-adapted middle rhodopsin as studied by solid-state nuclear magnetic resonance spectroscopy.

Middle rhodopsin (MR) found from the archaeon Haloquadratum walsbyi is evolutionarily located between two different types of rhodopsins, bacteriorhodopsin (BR) and sensory rhodopsin II (SRII). Some isomers of the chromophore retinal and the photochemical reaction of MR are markedly different from those of BR and SRII. In this study, to obtain the structural information regarding its active center (i.e., retinal), we subjected MR embedded in lipid bilayers to solid-state magic-angle spinning nuclear magnetic resonance (NMR) spectroscopy. The analysis of the isotropic 13C chemical shifts of the retinal chromophore revealed the presence of three types of retinal configurations of dark-adapted MR: (13-trans, 15-anti (all-trans)), (13-cis, 15-syn), and 11-cis isomers. The higher field resonance of the 20-C methyl carbon in the all-trans retinal suggested that Trp182 in MR has an orientation that is different from that in other microbial rhodopsins, owing to the changes in steric hindrance associated with the 20-C methyl group in retinal. 13Cζ signals of Tyr185 in MR for all-trans and 13-cis, 15-syn isomers were discretely observed, representing the difference in the hydrogen bond strength of Tyr185. Further, 15N NMR analysis of the protonated Schiff base corresponding to the all-trans and 13-cis, 15-syn isomers in MR showed a strong electrostatic interaction with the counter ion. Therefore, the resulting structural information exhibited the property of stable retinal conformations of dark-adapted MR.

On-Demand Chirality Transfer of Human Serum Albumin to Bis(thiophen-2-yl)hexafluorocyclopentenes through Their Photochromic Ring Closure.

Photochromic 1,2-bis(5-carboxy-3-methyl-2-thienyl)hexafluorocyclopentene and its dimethyl ester incorporated in human serum albumin (HSA) showed highly enantioselective photochromic ring-closing reactions upon 366 nm light irradiation. The absolute stereochemistry of the major ring-closed form of the dicarboxylic acid at the newly formed sp3 carbon atoms was determined to be (S,S) by the process of docking simulation of the diarylethene molecule and HSA followed by molecular dynamics calculations and comparison of the measured and calculated CD spectra. Esterification of the major closed form of the diacid gave the minor closed form of the diester. The absolute stereochemistry of the major diester was thus determined to be (R,R).

Vibrational circular dichroism of d-amino acid-containing peptide NdWFamide in the crystal form.

Microcrystals of l-Asn-d-Trp-l-Phe-NH2 (NdWFamide), a tripeptide derived from Aplysia kurodai that exhibits invertebrate cardiac activity, were evaluated by vibrational circular dichroism (VCD). The chirality of the tryptophan residue at the second position in NdWFamide was associated with the conformation and biological characteristics. The VCD spectrum of NdWFamide was a mirror image of its enantiomer; however, it was significantly different from that of its diastereomer, NWFamide, which is its precursor. The obtained VCD signals of NdWFamide were in good agreement with the VCD signals that were calculated based on the optimized aggregates of NdWFamide, which formed a helical-like backbone conformation. The evaluation of the VCD results revealed the conformation of NdWFamide in the crystalline state and succeeded in distinguishing its stereoisomers. Therefore, this study demonstrates VCD as a useful method for the structural analysis of naturally occurring d-amino acid-containing peptides.

Transport Properties of Flexible Composite Electrolytes Composed of Li1.5Al0.5Ti1.5(PO4)3 and a Poly(vinylidene fluoride-co-hexafluoropropylene) Gel Containing a Highly Concentrated Li[N(SO2CF3)2]/Sulfolane Electrolyte.

Flexible solid-state electrolyte membranes are beneficial for feasible construction of solid-state batteries. In this study, a flexible composite electrolyte was prepared by combining a Li+-ion-conducting solid electrolyte Li1.5Al0.5Ti1.5(PO4)3 (LATP) and a poly(vinylidene fluoride-co-hexafluoropropylene) (PVDF-HFP) gel containing a highly concentrated electrolyte of Li[N(SO2CF3)2] (LiTFSA)/sulfolane using a solution casting method. We successfully demonstrated the operation of Li/LiCoO2 cells with the composite electrolyte; however, the rate capability of the cell degraded with increasing LATP content. We investigated the Li-ion transport properties of the composite electrolyte and found that the gel formed a continuous phase in the composite electrolyte and Li-ion conduction mainly occurred in the gel phase. Solid-state 6Li magic-angle spinning NMR measurements for LATP treated with the 6LiTFSA/sulfolane electrolyte suggested that the Li+-ion exchange occurred at the interface between LATP and 6LiTFSA/sulfolane. However, the kinetics of Li+ transfer at the interface between LATP and the PVDF-HFP gel was relatively slow. The interfacial resistance of LATP/gel was evaluated to be 67 Ω·cm2 at 30 °C, and the activation energy for interfacial Li+ transfer was 39 kJ mol-1. The large interfacial resistance caused the less contribution of LATP particles to the Li-ion conduction in the composite electrolyte.