Reversible Activation of pH-Responsive Cell-Penetrating Peptides in Model Cell Membrane Relies on the Nature of Lipid

The pH response of pH-responsive cell-penetrating peptides in cell membrane is directly associated with many potential applications and cell activities such as drug delivery, membrane fusion, and protein folding, but it is still poorly understood. In this study, we used GALA as a model and applied sum frequency generation vibrational spectroscopy to systematically investigate the pH response of GALA in lipid bilayers with different hydrophobic length and lipid head groups. We determined the GALA structures in lipid bilayers by combining second-ordered amide I and amide III spectral signals, which can accurately differentiate the loop and α-helical structures at the interface. It is found that GALA can insert into fluid-phase lipid bilayers even at neutral pH, while lies down on the gel-phase lipid bilayer surface. Under acidic conditions, GALA inserts into both fluid-phase and gel-phase lipid bilayers. GALA adopts a mixed loop and α-helical structures in lipid bilayers. Besides, the reversible activation of GALA in lipid bilayers depends on the nature of lipid. After membrane insertion, GALA exits from the negative phosphoglycerol and positive ethylphosphocholine lipid bilayers at neutral pH, while it does not move out from the zwitterionic phosphocholine lipid bilayers. These findings will help us to understand how to enhance the efficacy of drug/gene delivery in cell membrane

Observing Peptide-Induced Lipid Accumulation in a Single-Component Zwitterionic Lipid Bilayer

Membrane domain formation plays a key role in various cellular functions and biological events. Lateral accumulation of lipids and proteins in biological membranes is one of the most important factors that control the domain formation. However, compared to numerous reports on the lipid aggregation or accumulation formed in the membranes composed of multiple components of lipids and cholesterol, the lipid accumulation in one-component phospholipid bilayer system is still rare. In this study, we demonstrate that short peptides can induce the lipid accumulation in a single-component zwitterionic lipid bilayer. By investigating the interaction between a short peptide of mastoparan (MP, a G-protein-activating peptide) and neutral phosphocholine lipid bilayers using sum frequency generation vibrational spectroscopy (SFG-VS), we have found that MP can cause a local accumulation of lipid molecules at the outer leaflet of the lipid bilayer, resulting in more than 10 times intensity increase in the signals from the CD₃ vibrational modes with respect to that of lipid monolayer at the air surface. We have validated that the lipid accumulation behavior originates from a specific hydrophobic-mismatching interaction in which the peptide is too short to span the lipid bilayer. Our results suggest that other mechanisms that do not involve perforation exist for the interactions between peptides and membranes. This finding broadens the range of systems and our basic understanding on lipid accumulation

Amide I SFG Spectral Line Width Probes the Lipid–Peptide and Peptide–Peptide Interactions at Cell Membrane In Situ and in Real Time

The balance of lipid–peptide and peptide–peptide interactions at cell membrane is essential to a large variety of cellular processes. In this study, we have experimentally demonstrated for the first time that sum frequency generation vibrational spectroscopy can be used to probe the peptide–peptide and lipid–peptide interactions in cell membrane in situ and in real time by determination of the line width of amide I band of protein backbone. Using a “benchmark” model of α-helical WALP23, it is found that the dominated lipid–peptide interaction causes a narrow line width of the amide I band, whereas the peptide–peptide interaction can markedly broaden the line width. When WALP23 molecules insert into the lipid bilayer, a quite narrow line width of the amide I band is observed because of the lipid–peptide interaction. In contrast, when the peptide lies down on the bilayer surface, the line width of amide I band becomes very broad owing to the peptide–peptide interaction. In terms of the real-time change in the line width, the transition from peptide–peptide interaction to lipid–peptide interaction is monitored during the insertion of WALP23 into 1,2-dipalmitoyl-<i>sn</i>-glycero-3-phospho-(1′-<i>rac</i>-glycerol) (DPPG) lipid bilayer. The dephasing time of a pure α-helical WALP23 in 1-palmitoyl-2-oleoyl-<i>sn</i>-glycero-3-phospho-(1′-<i>rac</i>-glycerol) and DPPG bilayer is determined to be 2.2 and 0.64 ps, respectively. The peptide–peptide interaction can largely accelerate the dephasing time

Ca²⁺ Ion Responsive Pickering Emulsions Stabilized by PSSMA Nanoaggregates

A novel Ca²⁺ ion responsive particulate emulsifier, which is based on copolymer nanoaggregates, is reported in this work. Results from dynamic light scattering (DLS) and cryo-transmission electron microscopy (cryo-TEM) indicate that the formation of poly (4-styrenesulfonic acid-<i>co</i>-maleic acid) sodium salt (PSSMA) nanoaggregates is strongly dependent on Ca²⁺ concentration. The PSSMA copolymer only aggregates above a critical Ca²⁺ concentration (0.2 M) with an average diameter of 10–40 nm. After dilution with water, PSSMA nanoaggregates are rapidly redissolved again. On the basis of the properties of PSSMA nanoaggregates, Ca²⁺ ion responsive Pickering emulsions were successfully prepared. At high Ca²⁺ concentrations, the emulsions with high stability against coalescence can be prepared with the size in the submicrometer range as determined by DLS. Cryo-TEM and dynamic interfacial tension results confirm the adsorption of PSSMA nanoaggregates at the interface, which is the key to the stability of the emulsions. More importantly, rapid demulsification can be achieved by dilution with water on demand. It is because, upon dilution with water, PSSMA nanoaggregates undergo a transition from stable nanoaggregates to individual polymer chains, which leads to interfacial desorption of nanoaggregates and rapid demulsification of emulsions. Thus, this finding presents a new manipulation on emulsion stability and is expected to provide a useful guidance in the fields of oil recovery, food science, environment protection, and so on

Columnar Self-Assemblies of Triarylamines as Scaffolds for Artificial Biomimetic Channels for Ion and for Water Transport

Triarylamine molecules appended with crown-ethers or carboxylic moieties form self-assembled supramolecular channels within lipid bilayers. Fluorescence assays and voltage clamp studies reveal that the self-assemblies incorporating the crown ethers work as single channels for the selective transport of K⁺ or Rb⁺. The X-ray crystallographic structures confirm the mutual columnar self-assembly of triarylamines and crown-ethers. The dimensional fit of K⁺ cations within the 18-crown-6 leads to a partial dehydration and to the formation of alternating K⁺ cation-water wires within the channel. This original type of organization may be regarded as a biomimetic alternative of columnar K⁺-water wires observed for the natural KcsA channel. Supramolecular columnar arrangement was also shown for the triarylamine-carboxylic acid conjugate. In this latter case, stopped-flow light scattering analysis reveals the transport of water across lipid bilayer membranes with a relative water permeability as high as 17 μm s^–1

Columnar Self-Assemblies of Triarylamines as Scaffolds for Artificial Biomimetic Channels for Ion and for Water Transport

Triarylamine molecules appended with crown-ethers or carboxylic moieties form self-assembled supramolecular channels within lipid bilayers. Fluorescence assays and voltage clamp studies reveal that the self-assemblies incorporating the crown ethers work as single channels for the selective transport of K⁺ or Rb⁺. The X-ray crystallographic structures confirm the mutual columnar self-assembly of triarylamines and crown-ethers. The dimensional fit of K⁺ cations within the 18-crown-6 leads to a partial dehydration and to the formation of alternating K⁺ cation-water wires within the channel. This original type of organization may be regarded as a biomimetic alternative of columnar K⁺-water wires observed for the natural KcsA channel. Supramolecular columnar arrangement was also shown for the triarylamine-carboxylic acid conjugate. In this latter case, stopped-flow light scattering analysis reveals the transport of water across lipid bilayer membranes with a relative water permeability as high as 17 μm s^–1

Columnar Self-Assemblies of Triarylamines as Scaffolds for Artificial Biomimetic Channels for Ion and for Water Transport

Triarylamine molecules appended with crown-ethers or carboxylic moieties form self-assembled supramolecular channels within lipid bilayers. Fluorescence assays and voltage clamp studies reveal that the self-assemblies incorporating the crown ethers work as single channels for the selective transport of K⁺ or Rb⁺. The X-ray crystallographic structures confirm the mutual columnar self-assembly of triarylamines and crown-ethers. The dimensional fit of K⁺ cations within the 18-crown-6 leads to a partial dehydration and to the formation of alternating K⁺ cation-water wires within the channel. This original type of organization may be regarded as a biomimetic alternative of columnar K⁺-water wires observed for the natural KcsA channel. Supramolecular columnar arrangement was also shown for the triarylamine-carboxylic acid conjugate. In this latter case, stopped-flow light scattering analysis reveals the transport of water across lipid bilayer membranes with a relative water permeability as high as 17 μm s^–1

ActD induces 45S rDNA fragility in ryegrass, maize, barley, rice and sorghum revealed by FISH.

<p>(A) Metaphase chromosome spreads revealed aberrant 45S rDNA phenotypes induced by ActD. The 45S rDNA FISH signal was the dense spot on chromosomes in untreated plants. In contrast, ActD-treated spreads exhibited highly stretched strands of rDNA signals or breaks on chromosomes after treatment with 15 µg/ml ActD. Bar = 5 µm. (B) Examples of fragile 45S rDNA phenotypes Bar = 5 µm. (C) Percentages of metaphase chromosome spreads with 45S rDNA lesions after treatment without or with 5 µg/ml and 15 µg/ml ActD, respectively. Number of evaluated spreads in each group was 300. (D) ActD treatment caused aberrant 45S rDNA signal patterns in nuclei. The interphase nuclei contained compact spot signals in normal plants whereas a mass of “beads-on-a-string” fibers were observed throughout the nucleoplasm in interphase nuclei after treatment with 15 µg/ml ActD. Bar = 10 µm. (E) Percentages of interphase nuclei with decondensed 45S rDNA fiber signals after treatment without or with 5 µg/ml and 15 µg/ml ActD, respectively. Number of evaluated nuclei in each group was 500.</p

Analysis of DNA methylation and histone modifications of 45S rDNA regions in maize.

<p>(A) Schematic representation of the maize rRNA genes and the positions of analyzed amplicons. (B) ActD treatment induced site-specific hypomethylation within the 45S rDNA promoter. The y-axis indicated the ratio of the clones with methylation sites and the x-axis indicated positions of CpG dinucleotides from −113 to +157. (C) ChIP analysis showed that the total level of histone H3 within 45S rDNA regions was decreased after treatment with 15 µg/ml ActD. DNA associated with histone H3 was immunoprecipitated with the anti-H3 antibody and primers specific for different region of 45S rDNAs were used to amplify DNA for quantitative real-time PCR. The y-axis values were the relative quantities of DNA and the x-axis indicated different regions of 45S rDNAs. Each experiment was repeated three times and the average value was shown with the SD. (D) ChIP analysis of levels of H3K9ac, H3K9me2, H3K4me2, H4K5ac and H4K16ac within 45S rDNA regions in maize after treatment without or with 15 µg/ml ActD. DNA associated with different histone modifications was immunoprecipitated with the related antibody and primers specific for different regions of the 45S rDNA were used to amplify DNA for quantitative real-time PCR. The y-axis indicated the ratio of the relative quantities of DNA in maize with ActD treatment to the relative quantities of DNA in maize without ActD treatment. Relative values were normalized to those of the total H3.The x-axis indicated different regions of 45S rDNAs. Each experiment was repeated three times and the average value was shown with the SD.</p

Primers used for quantitative analysis and DNA methylation analysis.

<p>Primers used for quantitative analysis and DNA methylation analysis.</p