Fabrication of Langmuir-Blodgett film of a fullerene derivative with a cyclic peptide as an anchor

A novel cyclic octapeptide carrying a fullerene unit and poly(ethylene glycol) at the side chain (cyclo8-C 60 + PEG) was synthesized, and its monolayer formation at the air/water interface and on a substrate was studied. Surface pressure-area per molecule isotherms indicated that cyclo8-C 60 + PEG formed a stable monolayer at the air/water interface. The cyclo8-C 60 + PEG monolayers prepared from various spreading volumes (i.e., from various initial areas per molecule) overlapped nicely on a single curve, suggesting that the molecules were uniformly dispersed on the surface without aggregation of the fullerene units. The uniform dispersibility is due to the scaffold effect of the cyclic peptide unit to keep the fullerene units away from each other. The formed monolayer could be quantitatively transferred onto a solid substrate. UV-vis absorption spectroscopy of the Langmuir-Blodgett (LB) monolayer showed that the electronic structure of the fullerene unit was not affected by the formation of the monolayer. Cyclic voltammetry of the LB monolayer in an aqueous solution containing redox species indicated that the LB monolayer was densely packed. Furthermore, reversible redox peaks attributed to the one-electron reduction of the fullerene unit were observed, showing that the redox property of the fullerene unit was also retained in the monolayer. It is thus concluded that the cyclic peptide is a good candidate as a scaffold for stable monolayer formation at the air/water interface and for intact immobilization of the fullerene moiety onto a substrate.     

Architecture of Peptide Assembly in Liposome

Abstract

Two chains of amphiphilic α-helical dodecapeptides were connected to a cyclic octapeptide in a parallel or antiparallel arrangement. Conformation, orientation, and association of the dodeca-peptide chains in the presence of dimyristoylphosphatidylcholine (DMPC) liposome were studied. The peptides were easily incorporated into the bilayer membrane. CD measurements revealed that the dodecapeptides took a distorted a-helix conformation due to intramolecular association. Fluorescence quenching measurement of a probe connected to each terminal region of the dodecapeptides revealed that the dodecapeptide chain was located on the membrane surface with a parallel orientation to the surface. Thermal denaturation of the helical structure was suppressed by connecting the two dodecapeptide chains to the cyclic peptide. These experimental results indicate that a supersecondary structure composed of associated α-helices was constructed on the surface of DMPC liposome by using a cyclic peptide as a template.     

Dragonfly Erythemis simplicicollis contains a novel adipokinetic neuropeptide

We have isolated a novel member of the adipokinetic hormone family of peptides from a methanolic extract of corpora cardiaca of the libellulid dragonfly Erythemis simplicicollis by using a single‐step reversed‐phase high performance liquid chromatography method and monitoring biological activity in various heterologous bioassays and a homologous one. The sequence, as determined by Edman degradation and mass spectrometry, was of an uncharged blocked octapeptide: pGlu‐Leu‐Asn‐Phe‐Thr‐Pro‐Ser‐Trp amide. The structure was confirmed by chemical synthesis. The synthetic peptide increased hemolymph lipids in the dragonfly and was active in another libellulid (Orthetrum julia‐falsum) as well, but to a lesser extent than the conspecific peptide Lia‐AKH, which is an isoform of the novel peptide differing by a Val (instead of Leu) at position 2. Since lipids are apparently used as substrate for muscle contraction during flight of Erythemis simplicicollis and the native peptide induces lipid mobilization, this novel peptide is denoted Ers‐AKH. Arch. Insect Biochem. Physiol. 40:99–106, 1999. © 1999 Wiley‐Liss, Inc.     

Self-assembly effect during the adsorption of polynucleotides on stearic acid langmuir-blodgett monolayer

Interaction of polyadenylic acid, poly(A), with stearic acid Langmuir-Blodgett (LB) monolayer was studied in different electrolyte surroundings. For this purpose LB films of stearic acid, transferred on the mica substrate from poly(A) containing subphase, were analyzed with atomic force microscopy (AFM). The density of polynucleotides surface coverage is ruled by the monovalent electrolyte concentration in the subphase that is in good agreement with previous results. Divalent cations in the subphase are needed to stabilize poly(A) molecules on the surface through formation of "salt bridges". At the very low divalent electrolyte concentration polynucleotides adsorb on the LB film to domains in which the effect of self-assembly is observed. Increase of divalent electrolyte concentration leads to the loss of this orientation effect. The explanation of this effect is proposed.     

Patterning of photosensitive polyimide LB film and its application in the fabrication of biomolecular microphotodiode array

Ultra thin film of photosensitive polyimide having benzene and sulfonyloxyimide moieties in the main chain was prepared using a Langmuir-Blodgett (LB) technique, and then micro array pattern of the polyimide LB film on a gold substrate was obtained by deep UV lithographic technique. In order to array cytochrome c molecules along the micro-patterned gold substrate, the well-characterized monolayer of cytochrome c was immobilized with a mixed monolayer of 11-mercaptoundecanoic acid (11-MUDA) and decanethiol. The redox activity and electron transfer between cytochrome c molecular center and gold electrode interface for the self-assembled cytochrome c monolayer were investigated by cyclic voltammetry measurement. Biomolecular photodiode consisting of cytochrome c and green fluorescent protein (GFP) onto the patterned gold substrate was fabricated by self-assembly process. The integration and morphology of cytochrome c and GFP were studied from the measurements of atomic force microscopy (AFM) and fluorescence emission. Especially, current-voltage characteristics of the protein multilayers were investigated by scanning tunneling microscopy (STM) and its application in biomolecular photodiode was also examined.     

Fine‐tuning sortase‐mediated immobilization of protein layers on surfaces using sequential deprotection and coupling

Increasing interest in protein immobilization on surfaces has heightened the need for techniques enabling layer‐by‐layer protein attachment. Here, we report a technique for controlling enzyme‐mediated immobilization of layers of protein on the surface using a genetically encoded protecting group. An enterokinase‐cleavable peptide sequence was inserted at the N‐terminus of bifunctional fluorescent proteins containing Sortase A substrate recognition tags at both ends to control Sortase A‐mediated protein immobilization on the surface layer‐by‐layer. Efficient, sequential immobilization of a second layer of protein using Sortase A required removal of the N‐terminal protecting group, suggesting the method enables multilayer synthesis using cyclic deprotection and coupling steps. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:824–831, 2017     

Redox-active cellulose Langmuir-Blodgett films containing beta-carotene as a molecular wire

Redox-active Langmuir-Blodgett (LB) films containing dihydrophytyl ferrocenoate (DFc) and beta-carotene (betaC) were fabricated by use of 6-O-dihydrophytylcellulose (DHPC) as a matrix. A mixture of DFc-DHPC formed a stable monolayer. Atomic force microscopy images revealed that the DFc molecules were dispersed uniformly throughout the surface in the ratio DFc:DHPC = 2:8 at 30 mN m-1. The DFc-DHPC monolayer was transferred successfully onto a substrate, yielding Y-type LB films. Cyclic voltammograms for the DFc-DHPC LB films on an indium tin oxide (ITO) electrode exhibited a well-defined surface wave. The voltammograms of the DFc-DHPC LB films exhibited 60-40% redox-active ferrocene moieties, whereas those of the DFc-DHPC-betaC LB films exhibited 90-70%. X-ray diffraction patterns indicated that the distance between layers was independent of betaC molecules incorporated into the LB films. Consequently, these results suggested that betaC can function as a molecular wire.     

Direct electrochemistry of hemoglobin immobilized on gold electrode by Langmuir-Blodgett technique

In this research, we reported a novel method of forming hemoglobin (Hb)-linoleic acid (LA) Langmuir-Blodgett (LB) monolayer by spreading Hb solution directly onto the subphase covered with a layer of LA. This method is suitable for preparing electrochemical devices with protein-lipid LB film because almost no protein adsorbed on electrode surface before protein-lipid film transferred from air-water interface to electrode, which ensured better electrode activity. The compressibility of Hb-LA monolayer was used to character the phase transition during compression process. Optimal experimental conditions were obtained by analyzing pressure-time, pressure-area and pressure-compressibility curves. The direct electrochemistry of Hb, which was immobilized on Au electrode surface incorporated with LA layer by LB method, was investigated using cyclic voltammetry for the first time. The electrode modified with Hb-LA LB film holds high electrochemical activity and shows a fast direct electron transfer of Hb. Redox peak currents increased linearly with the increase of scan rate, indicating a surface-controlled electrode process. The electron transfer rate constant was 2.68+/-0.45 s-1. As a target of this research, this work provides a new way to prepare biomimetic film and biosensor.     

The role of anti‐PAc (361–386) peptide SIgA antibody in professional oral hygiene of the elderly

Objective: Measurement of salivary IgA antibody (PAc‐peptide antibody, PPA) to amino acid residues 361–386 of Streptococcus mutans PAc, which possess a multiple binding motif to various HLA‐DR molecules and a B‐cell epitope that recognises the inhibiting antibody to S. mutans, is an indicator for the population numbers of mutans streptococci (MS) in human saliva. The purpose of this study was to clarify the role of PPA in infection control of MS after professional oral hygiene care. Materials and methods: Thirty‐nine dependently living institutionalised elderly subjects (75.9 ± 7.5 years; 10 males, 29 females) participated in the study. The measurements of PPA, MS, total streptococci (TS) and lactobacilli (LB) were performed by ELISA and culture techniques from saliva, plaque and tongue samples from the elderly. Results: After treatment using professional oral care, the numbers of MS decreased significantly at 6 months in saliva and tongue samples from the group not having PPA in comparison with the primary data; whereas in the PPA‐detected group, a significant decrease in MS number was shown immediately following professional care at 1–12 months in all samples. There was little difference in the numbers of LB at any of the time points. The numbers of TS decreased rapidly in PPA‐not detected group in comparison with the PPA‐detected group. Conclusion: PPA may be more effective for controlling MS number in the oral cavity after professional treatment. The measurement of PPA may be used for preventive instruction to dental caries at the chair side in the clinical setting.     

Partially induced transition from horizontal to vertical orientation of helical peptides at the air–water interface and the structure of their monolayers transferred on the solid substrates

To apply the Langmuir–Blodgett (LB) technique as a platform for investigating the fundamental properties of amphiphilic peptides (APs), we have investigated the structure of LB films using the APs. To vertically orient the helical APs like transmembrane proteins in the membrane, the primary structure of the APs was designed to have two domains: a hydrophilic domain (three amino acids) and a hydrophobic domain (ca. 20 amino acids). However, we are still far from having full control of their orientation. This study reports the contribution of the subphase temperature to the change in the orientation of helical APs. When the surface pressure–area isotherm of AP was observed at the subphase temperature at 41.5 °C, the isotherm exhibited a plateau, implying that a phase transition of the monolayer at the air–water interface occurred. Circular dichroism (CD) spectra of the monolayers transferred on the solid substrates revealed that the orientation of the helices changed at the pressure, where the plateau of the isotherm was observed. This change was not observed at 21.5 °C, i.e., the horizontal alignment of helixes was maintained. Atomic force microscopy (AFM) was used to systematically investigate the surface structure of the monolayers transferred at different surface pressures. A structural model of the monolayer that did not contradict with the results obtained by the three different techniques (the isotherm, CD spectroscopy, and AFM) was derived, and it was concluded that the horizontally oriented helices partially changed their orientation to vertical upon compression in the plateau region of the isotherm.     

A lead discovery strategy driven by a comprehensive analysis of proteases in the peptide substrate space

We present here a comprehensive analysis of proteases in the peptide substrate space and demonstrate its applicability for lead discovery. Aligned octapeptide substrates of 498 proteases taken from the MEROPS peptidase database were used for the in silico analysis. A multiple‐category naïve Bayes model, trained on the two‐dimensional chemical features of the substrates, was able to classify the substrates of 365 (73%) proteases and elucidate statistically significant chemical features for each of their specific substrate positions. The positional awareness of the method allows us to identify the most similar substrate positions between proteases. Our analysis reveals that proteases from different families, based on the traditional classification (aspartic, cysteine, serine, and metallo), could have substrates that differ at the cleavage site (P1–P1′) but are similar away from it. Caspase‐3 (cysteine protease) and granzyme B (serine protease) are previously known examples of cross‐family neighbors identified by this method. To assess whether peptide substrate similarity between unrelated proteases could reliably translate into the discovery of low molecular weight synthetic inhibitors, a lead discovery strategy was tested on two other cross‐family neighbors—namely cathepsin L2 and matrix metallo proteinase 9, and calpain 1 and pepsin A. For both these pairs, a naïve Bayes classifier model trained on inhibitors of one protease could successfully enrich those of its neighbor from a different family and vice versa, indicating that this approach could be prospectively applied to lead discovery for a novel protease target with no known synthetic inhibitors.     

Stabilisation of a short α‐helical VIP fragment by side chain to side chain cyclisation: a comparison of common cyclisation motifs by circular dichroism

A model octapeptide segment derived from vasoactive intestinal peptide (VIP) was utilised to investigate the effect of several conventional cyclisation methods on the α‐helical conformation in short peptide fragments. Three of the classical macrocyclisation techniques (i.e. lactamisation, ring‐closing metathesis and Huisgen cycloaddition) were applied, and the conformations of the resulting cyclic peptides, as well as their linear precursors, were compared by CD analysis. The visibly higher folding propensity of the triazole‐tethered peptide after azide‐alkyne CuAAC macrocyclisation illustrates that the secondary structure of a short peptide fragment can differ significantly depending on the chemical strategy used to covalently cross‐link side chain residues in a ‘helical’ fragment. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Langmuir-Blodgett films of a novel cellulose derivative with dihydrophytyl group: the ability to anchor beta-carotene molecules

A novel cellulose derivative, 6-O-dihydrophytylcellulose (DHPC), was first synthesized via a ring-opening polymerization and allowed to self-assemble onto an air-water interface. Langmuir-Blodgett (LB) films were characterized with atomic force microscope (AFM), UV-vis spectroscopy, and Fourier transform infrared spectroscopy. The surface pressure-area (pi-A) isotherms for DHPC and beta-carotene (betaC) mixture indicated strong interaction between these compounds to pack well. Thus, DHPC has the ability to anchor betaC in the monolayer. It was proved that a betaC-DHPC monolayer was transferred successfully onto a substrate, yielding Y-type LB films by UV spectroscopic analysis. The transmission and reflection-absorption IR spectra (RAS) indicated that the dihydrophytyl chains had almost trans-zigzag conformation and were oriented nearly perpendicular to the substrate. AFM section analysis revealed the thickness per layer to be 2.32 nm. Consequently, DHPC was found to be an appropriate matrix to fabricate the mixed LB films containing betaC.     

Assembly of binding loops on aromatic templates as VCAM‐1 mimetics

The design and synthesis of cyclic mimetics of VCAM‐1 protein that reproduce the integrin‐binding domain are presented. The unprotected peptide precursor 37 – 43 , Thr‐Gln‐Ile‐Asp‐Ser‐Pro‐Leu, was grafted onto functional templates of type naphthalene, biphenyl and benzyl through the chemoselective formation of C‐ and N‐terminal oximes resulting in a mixture of four isomeric forms due to syn–anti isomerism of the oxime bonds. Some isomers could be monitored by HPLC and identified by NMR. The molecule containing a naphthalene‐derived template was found to inhibit the VCAM‐1/VLA‐4 interaction more efficiently than previously reported for sulfur‐bridged cyclic peptides containing similar sequences. The finding confirms the importance of incorporating conformational constraints between the terminal ends of the peptide loop 37 – 43 in the design of synthetic inhibitors of the VCAM‐1/integrin interaction. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

Interactions of antimicrobial peptide from C-terminus of myotoxin II with phospholipid mono- and bilayers

Comparative studies of the effect of a short synthetic cationic peptide, pEM-2 (KKWRWWLKALAKK), derived from the C-terminus of myotoxin II from the venom of the snake Bothrops asper on phospholipid mono- and bilayers were performed by means of Langmuir Blodgett (LB) monolayer technique, atomic force microscopy and calcein leakage assay. Phospholipid mono- and bilayers composed of single zwitterionic or anionic phospholipids as well as lipid mixtures mimicking bacterial cell membrane were used. LB measurements indicate that the peptide binds to both anionic and zwitterionic phospholipid monolayers at low surface pressure but only to anionic at high surface pressure. Preferential interaction of the peptide with anionic phospholipid monolayer is also supported by a more pronounced change of the monolayer pressure/area isotherms induced by the peptide. AFM imaging reveals the presence of nanoscale aggregates in lipid/peptide mixture monolayers. At the same time, calcein leakage experiment demonstrated that pEM-2 induces stronger disruption of zwitterionic than anionic bilayers. Results of the study indicate that electrostatic interactions play a significant role in the initial recognition and binding of pEM-2 to the cell membrane. However, membrane rupturing activity of the peptide depends on interactions other than simple ionic attraction.     

Quantitative analysis of molecular orientation in chlorophyll a Langmuir monolayer: a polarized visible reflection spectroscopic study.

Polarized visible reflection spectra of a chlorophyll a (Chl.a) Langmuir monolayer have been measured in situ at various surface pressures. By applying Hansen's optics to the three-phase plane-bounded system (air/Chl.a monolayer/water), the negative reflection absorbances observed were reproduced satisfactorily by the theoretical calculation. Molecular orientation of Chl.a in the monolayer was evaluated quantitatively as a function of surface pressure, from the reflection absorbance of p- and s-polarized spectra of the red (Qy) band. It has been proven that Chl.a molecules in the monolayer form aggregates (islands) even in the low surface pressure region and that during the monolayer compression the molecules are gradually reorganized from inhomogeneous islands to ordered structures, with the chromophores oriented on the average vertically to the water surface.     

Directed self‐immobilization of alkaline phosphatase on micro‐patterned substrates via genetically fused metal‐binding peptide

Current biotechnological applications such as biosensors, protein arrays, and microchips require oriented immobilization of enzymes. The characteristics of recognition, self‐assembly and ease of genetic manipulation make inorganic binding peptides an ideal molecular tool for site‐specific enzyme immobilization. Herein, we demonstrate the utilization of gold binding peptide (GBP1) as a molecular linker genetically fused to alkaline phosphatase (AP) and immobilized on gold substrate. Multiple tandem repeats (n = 5, 6, 7, 9) of gold binding peptide were fused to N‐terminus of AP (nGBP1‐AP) and the enzymes were expressed in E. coli cells. The binding and enzymatic activities of the bi‐functional fusion constructs were analyzed using quartz crystal microbalance spectroscopy and biochemical assays. Among the multiple‐repeat constructs, 5GBP1‐AP displayed the best bi‐functional activity and, therefore, was chosen for self‐immobilization studies. Adsorption and assembly properties of the fusion enzyme, 5GBP1‐AP, were studied via surface plasmon resonance spectroscopy and atomic force microscopy. We demonstrated self‐immobilization of the bi‐functional enzyme on micro‐patterned substrates where genetically linked 5GBP1‐AP displayed higher enzymatic activity per area compared to that of AP. Our results demonstrate the promising use of inorganic binding peptides as site‐specific molecular linkers for oriented enzyme immobilization with retained activity. Directed assembly of proteins on solids using genetically fused specific inorganic‐binding peptides has a potential utility in a wide range of biosensing and bioconversion processes. Biotechnol. Bioeng. 2009;103: 696–705. © 2009 Wiley Periodicals, Inc.     

Intramolecular cyclization of the antimicrobial peptide Polybia‐MPI with triazole stapling: influence on stability and bioactivity

Cationic antimicrobial peptides have attracted increasing attention as a novel class of antibiotics to treat infectious diseases caused by pathogenic bacteria. However, susceptibility to protease is a shortcoming in their development. Cyclization is one approach to increase the proteolytic resistance of peptides. Therefore, to improve the proteolytic resistance of Polybia‐MPI, we have synthesized the MPI cyclic analogs C‐MPI‐1 (i‐to‐i+4) and C‐MPI‐2 (i‐to‐i+6) by copper(I)‐catalyzed azide–alkyne cycloaddition. Compared with MPI, C‐MPI‐1 displayed sustained antimicrobial activity and had enhanced anti‐trypsin resistance, while C‐MPI‐2 displayed no antimicrobial activity. The relationship between peptide structure and bioactivity was further investigated by probing the secondary structure of the peptides by circular dichroism. This showed that C‐MPI‐1 adopted an α‐helical structure in aqueous solution and, interestingly, had increased α‐helical conformation in 30 mM sodium dodecyl sulfate and 50% trifluoroethyl alcohol compared with MPI. C‐MPI‐2 that was not α‐helical in structure, suggesting that the propensity for α‐helix conformation may play an important role in cyclic peptide design. In addition, scanning electron microscopy, propidium iodide uptake, and membrane permeabilization assays indicated that MPI and the optimized analog C‐MPI‐1 had membrane‐active action modes, indicating that the peptides would not be susceptible to conventional resistance mechanisms. Our study provides additional insight into the influence of intramolecular cyclization at various positions on peptide structure and biological activity. In conclusion, the design and synthesis of cyclic analogs via click chemistry offer a new strategy for the development of stable antimicrobial agents. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.     

Modulation of protein–ligand interactions by photocleavage of a cyclic peptide using phosphatidylinositol 3‐kinase SH3 domain as model system

To photomodulate the interaction of the phosphatidylinositol 3‐kinase SH3 domain with a peptide ligand, a cyclic peptide (cyclic‐1) with a photolabile side chain‐to‐side chain linker was synthesized. The conformation of cyclic‐1 differs from that of the parent linear peptide, but becomes identical by UV‐irradiation. Accordingly, the binding affinity of cyclic‐1 to the SH3 domain increased upon conversion of the cyclic to a linear flexible structure by irradiation (K_d: 3.4 ± 1.7 and 0.9 ± 0.3 mM , respectively). These results confirm the usefulness of a photocleavable peptide for photocontrol of peptide–protein interactions. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

The use of a proteinaceous “cushion” with a polystyrene‐binding peptide tag to control the orientation and function of a target peptide adsorbed to a hydrophilic polystyrene surface

In immobilizing target biomolecules on a solid surface, it is essential (i) to orient the target moiety in a preferred direction and (ii) to avoid unwanted interactions of the target moiety including with the solid surface. The preferred orientation of the target moiety can be achieved by genetic conjugation of an affinity peptide tag specific to the immobilization surface. Herein, we report on a strategy for reducing the extent of direct interaction between the target moiety and surface in the immobilization of hexahistidine peptide (6His) and green fluorescent protein (GFP) on a hydrophilic polystyrene (PS) surface: Ribonuclease HII from Thermococcus kodakaraensis (cHII) was genetically inserted as a “cushion” between the PS‐affinity peptide tag and target moiety. The insertion of a cushion protein resulted in a considerably stronger immobilization of target biomolecules compared to conjugation with only a PS affinity peptide tag, resulting in a substantially enhanced accessibility of the detection antibody to the target 6His peptide. The fluorescent intensity of the GFP moiety was decreased by approximately 30% as the result of fusion with cHII and the PS‐affinity peptide tag but was fully retained in the immobilization on the PS surface irrespective of the increased binding force. Furthermore, the fusion of cHII did not impair the stability of the target GFP moiety. Accordingly, the use of a proteinaceous cushion appears to be promising for the immobilization of functional biomolecules on a solid surface. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:527–534, 2016