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The surface of microporous Norit® GAC 830 W carbon was fluoroalkylated by heating in 1,1,1,2-tetrafluoroethane and 1,1,1,2,2-pentafluoroethane at 400 and 500 °C. Elemental analysis, FTIR ATR spectroscopy, and TG studies showed the presence of ∼0.1–0.7 mmol g−1 of the fluorine-containing groups. Nitrogen adsorption measurements and quenched solid density functional theory simulations proved an increase in the mesoporosity. By combining the results of solid-state 19F NMR and XPS analyses, we determined the functionalization of the carbon surface with CF, CF3, and CF2 fluorine-containing groups. Supercapacitor electrodes prepared from these materials were subjected to the charge-discharge tests and electrical impedance measurements. During galvanostatic cycling in the aqueous 30% KOH, they showed an enhanced charge capacitance compared to the parent (830 W) and the reference (Supra 30) carbons. These observations suggest that the fluoroalkylated surface and the modified microporous structure have an additive effect on capacitance parameters of carbon electrodes studied in symmetric, negative and positive modes. The fluoroalkylation caused an increase in the surface capacitance up to 38%. Herein, we demonstrate the fluoroalkylation is a simple way to increase a capacitance of carbon electrodes.

Modulating the structural dynamics of biomembranes by inducing bilayer curvature and lipid packing defects has been highlighted as a practical tool to modify membrane-dependent cellular processes. Previously, we have reported on an amphipathic helical peptide derived from the N-terminal segment (residues 1-18, EpN18) of epsin-1, which can promote membrane remodeling including lipid packing defects in cell membranes. However, a high concentration is required to exhibit a pronounced effect. In this study, we demonstrate a significant increase in the membrane-remodeling effect of EpN18 by constructing a branched EpN18 homotrimer. Both monomer and trimer could enhance cell internalization of octaarginine (R8), a cell-penetrating peptide. The EpN18 trimer, however, promoted the uptake of R8 at an 80-fold lower concentration than the monomer. Analysis of the generalized polarization of a polarity-sensitive dye (di-4-ANEPPDHQ) revealed a higher efficacy of trimeric EpN18 in loosening the lipid packing in the cell membrane. Circular dichroism measurements in the presence of lipid vesicles showed that the EpN18 trimer has a higher α-helix content compared with the monomer. The stronger ability of the EpN18 trimer to impede negative bilayer curvature is also corroborated by solid-state 31P NMR spectroscopy. Hence, trimerizing peptides can be considered a promising approach for an exponential enhancement of their membrane-remodeling performance.

Trans -4,5-Difluoromethano-proline was incorporated into the cyclic antimicrobial peptide gramicidin S in place of a native proline residue. Introduction of this intrinsically unstable amino acid into the polypeptide backbone was achieved using a dipeptide strategy. The stable dipeptide building block with the N-acylated 4,5-difluoromethano-proline fragment was obtained by direct difluorocyclopropanation of an unsaturated precursor. The influence of the unnatural amino acid on the conformation and function of gramicidin S was evaluated using circular dichroism and biological assays. The application of trans -4,5-difluoromethano-proline as a new label for solid state 19 F NMR structure analysis of membrane-active peptides was tested on gramicidin S and compared with previous labeling schemes.

Prostatic acid phosphatase (PAP) is a protein abundantly present in human seminal fluid. PAP plays important role in fertilization. Its 39-amino-acid fragment, PAP(248–286), is effective in enhancing infectivity of HIV virus. In this work, we determined the spatial structure in aqueous solution of a heptapeptide within the PAP fragment, containing amino acid residues 266–272 (Glu-Ile-Leu-Asn-His-Met-Lys). We also report the structure of the complex formed by this heptapeptide with sodium dodecyl sulfate micelles, a model of a biological membrane, as determined by 1H NMR spectroscopy and 2D NMR (TOCSY, HSQC-HECADE, NOESY) spectroscopy. Complex formation was confirmed by chemical shift alterations in the 1H NMR spectra of the heptapeptide, as well as by the signs and values of NOE effects. We also present a comparison of the spatial structure of Glu-Ile-Leu-Asn-His-Met-Lys in water and in complex with sodium dodecyl sulfate.

Tat signal peptides provide the key signature for proteins that get exported by the bacterial twin arginine translocase. We have characterized the structure of the PhoD signal peptide from Bacillus subtilis in suitable membrane-mimicking environments. High-resolution 13C/15N NMR analysis in detergent micelles revealed a helical stretch in the signal peptide between positions 5 and 15, in good agreement with secondary structure prediction and circular dichroism results. This helix was found to be aligned parallel to the membrane surface according to oriented circular dichroism experiments carried out with planar lipid bilayers. The N-terminal α-helix exhibits a pronounced amphiphilic character, in contrast to the general view in the literature. So far, signal sequences had been supposed to consist of a positively charged N-terminal domain, followed by an α-helical hydrophobic segment, plus a C-terminal domain carrying the peptidase cleavage site. Based on our new structural insights, we propose a model for the folding and membrane interactions of the Tat signal sequence from PhoD.

An optimized procedure for the multigram synthesis of 3-(trifluoromethyl)bicyclo[1.1.1]pent-1-ylglycine (CF 3 -Bpg) has been developed. The overall yield of the synthesis for the optimized up-scaling was increased from 35% to 53%. Moreover, conditions for separating the key isomeric aminonitriles 7 and 8 by crystallization were found, which greatly facilitated the isolation of 8 on a multigram scale. Following this optimized protocol, 100 g of optically pure CF 3 -Bpg have been synthesized.

Oriented circular dichroism (OCD) was used to characterize and compare in a quantitative manner the secondary structure and concentration dependent realignment of the antimicrobial peptides PGLa and MSI-103, and of the structurally related cell-penetrating peptide MAP in aligned phospholipid bilayers. All these peptides adopt an amphiphilic alpha-helical conformation, and from solid-state NMR analysis they are known to bind to membranes in two distinct orientations depending on their concentration. At low peptide/lipid (P/L) ratio the helices are aligned parallel to membrane surface (S-state), but with increasing concentration they realign to a tilted orientation (T-state), getting immersed into the membrane with an oblique angle supposedly as a result of dimer-formation. In macroscopically aligned liquid crystalline 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine bilayers the two limiting states are represented by distinct OCD spectra, and all spectra at intermediate peptide concentrations can be described by a linear combination of these two line shapes. The corresponding fraction of molecules occupying the T-state was determined by fitting the intermediate spectra with a superposition of the two extreme line shapes. By plotting this fraction versus 1/(P/L), the threshold P/L* ratio for realignment was extracted for each of the three related peptides. Despite their structural similarity distinctly different thresholds were obtained, namely for MSI-103 realignment starts already at a low P/L of approximately 1:236, for a MAP derivative (using a nonaggregating analog containing a D-amino acid) the transition begins at P/L approximately 1:156, whereas PGLa needs the highest concentration to flip into T-state at P/L approximately 1:85. Analysis of the original MAP sequence (containing only L-amino acids) gave OCD spectra compatible with beta-pleated conformation, suggesting that this peptide starts to aggregate with increasing concentration, unlike the other helical peptides. All these changes in peptide conformation and membrane alignment observed here by OCD seem to be functionally relevant, as they can be correlated with the membrane perturbing activities of the three antimicrobial and cell-penetrating sequences.

Dipolar couplings contain information on internuclear distances as well as orientational constraints. To characterize the structure of the antimicrobial peptide gramicidin S when bound to model membranes, two rigid 4-CF 3 -phenylglycine labels were attached to the cyclic backbone such that they reflect the behavior of the entire peptide. By solid state 19 F NMR we measured the homonuclear dipolar couplings of the two trifluoromethyl-groups in oriented membrane samples. Using the CPMG experiment, both the strong couplings within each CF 3 -group as well as the weak coupling between the two CF 3 -groups could be detected. An intra -CF 3 -group dipolar coupling of 86 Hz and a weak inter -group coupling of 20 Hz were obtained by lineshape simulation of the complex dipolar spectrum. It is thus possible to explore the large distance range provided by 19 F-labels and to resolve weak dipolar couplings even in the presence of strong intra -CF 3 couplings. We applied this approach to distinguish and assign two epimers of the labeled gramicidin S peptide on the basis of their distinct 19 F dipolar coupling patterns.

The effects of five fragments of prostatic acid phosphatase on dimyristoylphosphatidylcholine lipid multi-lamellar liposomes were studied by 2H and 31P NMR spectroscopy and those on planar supported multi-bilayers of the same lipid, by 1H and 31P NMR spectro-scopy. It was found that hydrophobic interaction is a dominated factor of the peptide–membrane binding, while the short α-helical fragments PAP(262-270) and PAP(262-272) strongly interact with the membrane at the interface, generally following to the Gibbs free energy of water-to-interface insertion.

Solid-state 19F-NMR structure analysis of membrane-bound peptides is routinely feasible now in macroscopically oriented samples. For many long amphiphilic α-helices, their membrane-induced folding, re-alignment and insertion into the lipid bilayer has been characterized as a series of consecutive steps, ultimately leading to pore-formation across the membrane. More recently, however, we have examined various short peptides that are not long enough to span the lipid bilayer, and whose biological action cannot be explained in terms of classical models of oligomeric pore formation. For example, the 13-aa antimicrobial peptide Temporin A from frog skin, as well as the 11-aa antimicrobial and cell penetrating sequence of BP100, are found to insert as α-helices with an oblique tilt angle into membranes over a wide range of conditions. In this alignment the height of the backbone matches only the width of a single monolayer. Based on this structural knowledge and on fluorescence studies with lipid vesicles, and further supported by all-atom MD simulations, we postulate a novel proton shuttling mechanism for Temporin A, similar to the decoupler molecule CCCP. BP100, on the other hand, appears to be tautly suspended inbetween the polar bilayer surfaces of DMPC model membranes via snorkeling lysine residues, and the same unusual structure is observed by 19F-NMR even in native bacterial membranes.

Amphipathic membrane-active peptides (antimicrobial, hemolytic, cell-penetrating, fusogenic, etc.) achieve their functions by distinct interaction with lipid bilayers. Some typical structural modes are described in terms of models like the “barrel stave”, “toroidal pore”, “carpet” etc. These models are related to the alignment states of the peptides in the lipid bilayers (surface bound “S-state”, inserted “I-state” or tilted “T-state”), which can be readily characterized by solid state NMR. When determining such alignment, factors like peptide/lipid ratio, charge of the bilayer surface, thickness of the bilayer core, presence of cholesterol, and humidity are typically investigated. Yet, the lipid phase state as an explicit variable parameter has not received much attention so far. Here, we demonstrate that a change in the lipid phase can directly trigger the re-alignment of many peptides. Several representative examples are illustrated here: PGLa, PGLa/Magainin, gramicidin S, SAP and alamethicin. In macroscopically oriented DMPC bilayers, using highly-sensitive 19F-NMR we have monitored the changes between known alignment states of these peptides as a function of temperature, covering both the gel and liquid-crystalline states of DMPC. We show that for all peptides studied the alignment in the gel-state differs from the one in the liquid-crystalline bilayers and can be reversibly changed by passing through the lipid phase transition temperature. The relevance of these finding for the phase state of native biological membranes and interactions of membrane-active peptides with them will be discussed.