Channel current analysis estimates the pore-formation and the penetration of transmembrane peptides.

We measured the current signal of the transmembrane model peptides using the barrel-stave, toroidal pore, and penetration models in order to establish a precise assignment of the channel signals. In addition, we analyzed the spike signals to estimate the membrane penetration of model cell-penetration peptides of different lengths.

DNA G-Wire Formation Using an Artificial Peptide is Controlled by Protease Activity.

The development of a switching system for guanine nanowire (G-wire) formation by external signals is important for nanobiotechnological applications. Here, we demonstrate a DNA nanostructural switch (G-wire <--> particles) using a designed peptide and a protease. The peptide consists of a PNA sequence for inducing DNA to form DNA-PNA hybrid G-quadruplex structures, and a protease substrate sequence acting as a switching module that is dependent on the activity of a particular protease. Micro-scale analyses via TEM and AFM showed that G-rich DNA alone forms G-wires in the presence of Ca2+, and that the peptide disrupted this formation, resulting in the formation of particles. The addition of the protease and digestion of the peptide regenerated the G-wires. Macro-scale analyses by DLS, zeta potential, CD, and gel filtration were in agreement with the microscopic observations. These results imply that the secondary structure change (DNA G-quadruplex <--> DNA/PNA hybrid structure) induces a change in the well-formed nanostructure (G-wire <--> particles). Our findings demonstrate a control system for forming DNA G-wire structures dependent on protease activity using designed peptides. Such systems hold promise for regulating the formation of nanowire for various applications, including electronic circuits for use in nanobiotechnologies.

Elemental composition control of gold-titania nanocomposites by site-specific mineralization using artificial peptides and DNA.

Biomineralization, the precipitation of various inorganic compounds in biological systems, can be regulated in terms of the size, morphology, and crystal structure of these compounds by biomolecules such as proteins and peptides. However, it is difficult to construct complex inorganic nanostructures because they precipitate randomly in solution. Here, we report that the elemental composition of inorganic nanocomposites can be controlled by site-specific mineralization by changing the number of two inorganic-precipitating peptides bound to DNA. With a focus on gold and titania, we constructed a gold-titania photocatalyst that responds to visible light excitation. Both microscale and macroscale observations revealed that the elemental composition of this gold-titania nanocomposite can be controlled in several ten nm by changing the DNA length and the number of peptide binding sites on the DNA. Furthermore, photocatalytic activity and cell death induction effect under visible light (>450 nm) irradiation of the manufactured gold-titania nanocomposite was higher than that of commercial gold-titania and titania. Thus, we have succeeded in forming titania precipitates on a DNA terminus and gold precipitates site-specifically on double-stranded DNA as intended. Such nanometer-scale control of biomineralization represent a powerful and efficient tool for use in nanotechnology, electronics, ecology, medical science, and biotechnology.

Peptides for Silica Precipitation: Amino Acid Sequences for Directing Mineralization.

BACKGROUND: Peptides are promising compounds for use in inorganic or organic-inorganic hybrid syntheses (mineralization) and offer several advantages over proteins. Meanwhile, silica-based nanomaterials have been extensively investigated for many years because of their potential application in a diverse range of technologies, including catalysis, sensing, separation, enzyme immobilization, and gene and drug delivery. Considerable progress has been made over the past decade in understanding the molecular mechanisms underpinning biosilicification and the biomimetic synthesis of patterned nanosilica using peptides. OBJECTIVES: This mini-review focuses on various peptide sequences, especially short peptide sequences (30 residues or less), for silica mineralization. METHODS: We first briefly review early studies on silica mineralization using proteins to provide background information. This is followed by a discussion of promising peptide sequences and attempts to discern the relationship between amino acid sequence, their potential for mineralization, and the properties of the mineral product. RESULTS: The synthetic control of silica mineralization using engineered proteins, such as recombinant silicateins and silaffins, was inspired by silica biomineralization by natural proteins from organisms (sponges, diatoms, and plants). Concurrently, several papers described the utility of well-structured protein assemblies as templates for silica mineralization. These template-directed syntheses of well-structured silica deposits were first conducted using natural proteins or protein assemblies such as collagen fibers and virus hollow protein tubes. Then we reviewed a selection of short peptides (30 residues or less) that had been successfully used for silica mineralization. Almost all peptides developed to date can be sorted by classification like proteins (synthetic control of silica mineralization or utility of templates for silica mineralization): the first class of peptides is used for peptide-directed synthesis, and the second is used for template-directed synthesis after the peptides have assembled and formed nanostructure such as fibers and tubes. The presented peptides were classified and arranged according to the classification. Additionally, we briefly introduced silica mineralization triggered by the combination of short silica-precipitating peptides and template molecules. CONCLUSION: In this mini-review we focused on various peptide sequences, especially short peptide sequences of 30 residues or less, designed for silica mineralization. The peptides have been used both for peptide-directed silica mineralization and for template-directed silica mineralization. The recent advances in peptide-driven mineralization reviewed here suggest that it will soon be possible to completely control the silica mineralization process using peptides. Mineralization systems using peptides will provide researchers with new tools for controlling various inorganic syntheses and the production of organic-inorganic materials for nanobiochemistry and materials chemistry research.

Modification of the N-Terminus of a Calcium Carbonate Precipitating Peptide Affects Calcium Carbonate Mineralization.

BACKGROUND: A core sequence (the 9 C-terminal residues) of calcification-associated peptide (CAP- 1) isolated from the exoskeleton of the red swamp crayfish was previously shown to control calcium carbonate precipitation with chitin. In addition, a modified core sequence in which the phosphorylated serine at the N terminus is replaced with serine exhibits was also previously shown to alter precipitation characteristics with chitin. OBJECTIVES: We focused on calcium carbonate precipitation and attempted to elucidate aspects of the mechanism underlying mineralization. We attempted to evaluate in detail the effects of modifying the N-terminus in the core sequence on calcium carbonate mineralization without chitin. METHODS: The peptide modifications included phosphorylation, dephosphorylation, and a free or acetylated Nterminus. The peptides were synthesized manually on Wang resin using the DIPCI-DMAP method for the first residue, and Fmoc solid phase peptide synthesis with HBTU-HOBt for the subsequent residues. Prior to calcium carbonate precipitation, calcium carbonate was suspended in MilliQ water. Carbon dioxide gas was bubbled into the stirred suspension, then the remaining solid CaCO3 was removed by filtration. The concentration of calcium ions in the solution was determined by standard titration with ethylenediaminetetraacetate. Calcium carbonate precipitation was conducted in a micro tube for 3 h at 37°C. We used the micro-scale techniques AFM (atomic force microscopy) and TEM (transmission electron microscopy), and the macro-scale techniques chelate titration, HPLC, gel filtration, CD (circular dichroism) and DLS (dynamic light scattering). RESULTS: We determined the morphologies of the calcium carbonate deposits using AFM and TEM. The pS peptide provided the best control of the shape and size of the calcium carbonate round particles. The acetylated peptides (Ac-S and Ac-pS) provided bigger particles with various shapes. S peptide provided a mixture of bigger particles and amorphous particles. We verified these findings using DLS. All the peptide samples produced nanostructures of the expected size in agreement with the AFM and TEM results. We estimated the abilities of these peptides to precipitate calcium carbonate by determining the residual calcium hydrogen carbonate concentration by standard titration with ethylenediaminetetraacetate after calcium carbonate precipitation. The Ac-pS peptide showed the lowest residual calcium hydrogen carbonate concentration whereas the S peptide showed the highest, suggesting that the precipitating activities of these peptides towards calcium carbonate correlated with peptide net charge. Then the gel filtration results showed a large oligomer peak and a small oligomer/monomer peak for all peptide samples in agreement with the AFM, TEM and DLS results. CD measurements showed that all the peptides formed random-coil-like structures. Thus, we used both macro- and micro-observation techniques such as chelate titration, DLS, AFM and TEM to show that the calcium carbonate precipitating activities of four derivatives of the core sequence of CAP-1 may correlate with the peptide net charge. CONCLUSION: These peptides mainly act as a catalyst rather than as a binder or component of the calcium carbonate deposits (as a template). On the other hand, the morphologies of the calcium carbonate deposits appeared to be dependent on the ability of the peptide to assemble and act as a template. Consequently, elucidating the relationship between peptide sequence and the ability of the peptide to assemble would be indispensable for controlling precipitate morphologies in the near future. This knowledge would provide important clues for elucidating the relationship between peptide sequence and mineralization ability, including deposit morphology and precipitating activity, for use in nanobiochemistry and materials chemistry research.