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.

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.

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.

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.

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.

Structural determination of the sheath-forming polysaccharide of Sphaerotilus montanus using thiopeptidoglycan lyase which recognizes the 1,4 linkage between α-d-GalN and ß-d-GlcA.

Sphaerotilus natans is a filamentous sheath-forming bacterium commonly found in activated sludge. Its sheath is assembled from a thiolic glycoconjugate called thiopeptidoglycan. S. montanus ATCC-BAA-2725 is a sheath-forming member of stream biofilms, and its sheath is morphologically similar to that of S. natans. However, it exhibits heat susceptibility, which distinguishes it from the S. natans sheath. In this study, chemical composition and solid-state NMR analyses suggest that the S. montanus sheath is free of cysteine, indicating that disulfide linkage is not mandatory for sheath formation. The S. montanus sheath was successfully solubilized by N-acetylation, allowing solution-state NMR analysis to determine the sugar sequence. The sheath was susceptible to thiopeptidoglycan lyase prepared from the thiopeptidoglycan-assimilating bacterium, Paenibacillus koleovorans. The reducing ends of the enzymatic digests were labeled with 4-aminobenzoic acid ethyl ester, followed by HPLC. Two derivatives were detected, and their structures were determined. We found that the sheath has no peptides and is assembled as follows: [→4)-ß-d-GlcA-(1→4)-ß-d-Glc-(1→3)-ß-d-GalNAc-(1→4)-α-d-GalNAc-(1→4)-α-d-GalN-(1→]n (ß-d-Glc and α-d-GalNAc are stoichiometrically and substoichiometrically 3-O-acetylated, respectively). Thiopeptidoglycan lyase was thus confirmed to cleave the 1,4 linkage between α-d-GalN and ß-d-GlcA, regardless of the peptide moiety. Furthermore, vital fluorescent staining of the sheath demonstrated that elongation takes place at the tips, as with the S. natans sheath.

Multidimensional Vibrational Circular Dichroism Apparatus Equipped with Quantum Cascade Laser and Its Use for Investigating Some Peptide Systems Containing d-Amino Acids.

We developed a multidimensional vibrational circular dichroism system with a positional coordinate, making it possible to move in both x- and y-directions using an automatic stage. Quantum cascade laser (QCL) was used as a light source to achieve high intensity and narrow focusing. The QCL emitted light in the wavenumber range of 1500-1740 cm-1, which encompassed absorption bands assigned to the stretching vibrations of amides I and II. The operation of the instrument was analyzed for samples containing amide groups. An aqueous solution of Gly-l-Leu or Gly-d-Leu was measured under the background absorbance as high as 3.5 due to the water medium. The spectra were recorded by scanning at 1.0 cm-1 steps. The time required for performing measurement at each wavenumber was less than 1 s. The mirror-image relation was maintained between the optical antipodes. A peak assigned to amide II appeared clearly at around 1580 cm-1. In the case of KBr pellets containing the same compounds, peaks assigned to amide I and II were observed. For two-dimensional pattering, a KBr pellet comprising two domains of amino acids (or l-Ala and d-Ser) was investigated. The distribution of each component within the pellet was obtained under the two-dimensional alignment at the spatial interval of 2.5 mm.

Separation of D-amino acid-containing peptide phenylseptin using 3,3'-phenyl-1,1'-binaphthyl-18-crown-6-ether columns.

Several D-amino acid-containing peptides (DAACPs) with antimicrobial, cardio-excitatory, or neuronal activities have been found in several species. Here, we demonstrated the chiral separation of the antimicrobial peptide diastereomers, D-phenylseptin and L-phenylseptin using (S) and (R) 3,3'-phenyl-1,1'-binaphthyl-18-crown-6-ether columns (CR-I (+) and CR-I (-), respectively) and also investigated the underlying mechanism. First, using D-amino acid-containing tripeptide Phe-Phe-Phe-OH, we found that CR-I (+) could be used to recognize diastereomeric tripeptides containing an L-amino acid as the first residue. On the contrary, CR-I (-) enabled separation of a series of diastereomers with D-amino acid as the first residue. Therefore, we achieved separation of the stereoisomers using the chiral columns depending on the position of the D- amino acid in the peptide and demonstrated the orthogonality of separations of the chiral columns. Then, using CR-I (+), we separated amphibian antimicrobial peptide diastereomers, L- and D-phenylseptin, which have the sequences, L-Phe-L-Phe-L-Phe and L-Phe-D-Phe-L-Phe at their N-termini, respectively. In order to understand the host-guest interactions, we performed molecular dynamics simulations for L-Phe-L-Phe-L-Phe tripeptide-CR-I molecule complex systems. Three hydrogen bonds between the N-terminal amine group -NH3+ and the crown ether oxygens were the dominant interactions. The hydrophobic interactions between phenyl-rings in the chiral selector unit of CR-I (+) and the side chains of 2nd and 3rd residues of the peptide also contributed to the affinity. Our results show that the CR-I (+)-column can be applied for the separation of endogenous DAACPs generated by the post-translational modification.

Solid-State Nuclear Magnetic Resonance Structural Study of the Retinal-Binding Pocket in Sodium Ion Pump Rhodopsin.

The recently identified Krokinobacter rhodopsin 2 (KR2) functions as a light-driven sodium ion pump. The structure of the retinal-binding pocket of KR2 offers important insights into the mechanisms of KR2, which has motif of Asn112, Asp116, and Gln123 (NDQ) that is common among sodium ion pump rhodopsins but is unique among other microbial rhodopsins. Here we present solid-state nuclear magnetic resonance (NMR) characterization of retinal and functionally important residues in the vicinity of retinal in the ground state. We assigned chemical shifts of retinal C14 and C20 atoms, and Tyr218Cζ, Lys255Cε, and the protonated Schiff base of KR2 in lipid environments at acidic and neutral pH. 15N NMR signals of the protonated Schiff base showed a twist around the N-Cε bond under neutral conditions, compared with other microbial rhodopsins. These data indicated that the location of the counterion Asp116 is one helical pitch toward the cytoplasmic side. In acidic environments, the 15N Schiff base signal was shifted to a lower field, indicating that protonation of Asp116 induces reorientation during interactions between the Schiff base and Asp116. In addition, the Tyr218 residue in the vicinity of retinal formed a weak hydrogen bond with Asp251, a temporary Na+-binding site during the photocycle. These features may indicate unique mechanisms of sodium ion pumps.