Chemical synthesis of TASP arrays and their application in protein design

A combinatorial synthesis of de novo proteins is described. The concept of template-assembled synthetic proteins (TASP) has been adapted to an orthogonal assembly of small libraries of purified peptide building blocks. It is combined with the spot synthesis of peptides which is exploited to array cyclic decapeptide templates on cellulose membranes. A cleavable linker on the cellulose allows control of the synthesis. The hydrophilic proteins are constructed by successive cleavage of orthogonal protecting groups on the template, followed by coupling of amphipathic helices in a predefined orientation and finally by incorporation of a cofactor. Libraries of peptides with variation of the amino acids expected to be close to the cofactor were coupled to the cellulose-bound template in all combinations, yielding up to 500 variants of a protein. Cofactors have been inserted either at non-covalent binding sites as heme and Cu2+ or by covalent modification of amino acids as Ru-bipyridine or flavin. The proteins were screened by recording their UV-vis spectra directly on the solid support. The properties screened include the redox potential of heme proteins, charge transfer bands indicating the ligation of Cu-centers, enzymatic activity, and folding stability. Synthesis of the best hits as soluble variants was used for detailed characterization. Iterative improvement in a second screening cycle was efficient in finding novel copper proteins. We discuss the prospects of synthesizing proteins by extending the concept to beta-sandwich proteins and construction of efficient peptide libraries with computer-supported design, as well as the possible usage of improved solid phase materials.     

固体有机膜的湿透过度测量

膜法除湿具有装置体积小、重量轻和能耗低等特点。本文提供了一种新的测量膜透湿能力的方法,用改进的实验室释放分析系统(FLEC)对国产多孔固体高分子有机膜的透湿性能进行了测量,获得了不同孔径和厚度的尼龙膜、混合纤维素膜、醋酸纤维素膜和疏水PVDF膜的湿透过度,并对实验结果的误差进行了分析。结果表明,不论是憎水性膜还是亲水性膜,水蒸气均能显著透过,湿透过度与膜材料、膜孔径、厚度有关,小膜厚、大孔径以及强亲水性均有利于传湿。     

Track-etch membranes as templates enabled nano/micro technology: a review

Many techniques are being used in order to synthesize nano-micro materials falling under the realm of nanotechnology. It need not be overemphasized that the miniaturization of devices and synthesis of new materials have a tremendous role in the development of powerful electronics as well as material based technologies in other areas but for the laws of quantum mechanics posing limitations besides the increasing cost and difficulties in manufacturing in such a small scale. The quest, therefore, for the alternative technologies, have stimulated a surge of interest in nano-meter scale materials and devices in the recent years. Metallic as well as semiconducting nano wires are the most attractive materials because of their unique properties having myriad of applications like interconnects for nano-electronics, magnetic devices, chemical and biosensors, whereas the hollow tubules are equally considered to be candidates for more potent applications — both in physical as well as biosciences. Materials’ processing for nano-structured devices is indispensable to their rational design. The technique, known as “Template Synthesis”, using electrochemical-electro less deposition is one of the most important processes for manufacturing nano-micro structures, nano-composites and devices and is relatively inexpensive and simple. The technique involves using membranes — ion crafted ones (popularly known as Particle Track-Etch Membranes or Nuclear Track Filters), alumite substrate membranes, besides other types of membranes as templates. The parameters viz., diameter as well as length i.e., aspect ratio, shape and wall surface traits in these membranes are controllable. In the present article a detailed review of this technique using track-etch membranes as templates in synthesis of nano-micro materials including hybrid materials and devices like field-ion emitters, resonant tunneling diodes (RTDs) etc. is presented including most of the results obtained in our laboratory.      

Interactions of lactoferricin B derivatives with model cell membrane studied by Raman spectroscopy

In this work, we employed Raman spectroscopy to study the effect of the antimicrobial peptide lactoferricin B (LfB) on model cell membranes. We used two derivatives of LfB (RRWQWRMKKLG and RRWQWR) with broad‐spectrum activity against gram‐positive and gram‐negative bacteria, fungus, viruses and tumors. Raman spectra of the peptides showed no conformational change in the temperature range 4–60 °C. The positions of the amide I and amide III bands suggest that in an aqueous solution these peptides preferentially adopt a random coil‐like conformation. We also investigated the effect the peptides had on the melting behavior of model cell membranes composed of zwitterionic lipid dipalmitoylglycero‐phosphocholine (DPPC) and anionic lipid dipalmitoylglycero‐phosphoglycerol (DPPG). Raman CH stretching bands were used to follow the melting of the lipid vesicles. We found that the melting of DPPC lipid vesicles is not affected by the presence of the peptides, while the presence of the peptides reduced cooperativity of the phase transition for anionic DPPG vesicle, suggesting that both peptides interact strongly and specifically with this model cell membrane composed of anionic lipid. Copyright © 2006 John Wiley & Sons, Ltd.     

Development of high performance nano-porous polyethersulfone ultrafiltration membranes with hydrophilic surface and superior antifouling properties

Hydrophilic nano-porous polyethersulfone ultrafiltration membranes were developed for milk concentration. The membranes were prepared from new dope solution containing polyethersulfone (PES)/polyvinylpirrolidone (PVP)/polyethyleneglycole (PEG)/cellulose acetate phthalate (CAP)/acrylic acid/Triton X-100 using phase inversion induced by immersion precipitation technique. This casting solution leads to formation of new hydrophilic membranes. The morphological studies were investigated by scanning electron microscopy (SEM) and atomic force microscopy (AFM). In addition, the hydrophilicity and performance of membranes were examined by contact angel measurements and cross-flow filtration (pure water flux, milk water permeation, protein rejection and antifouling measurements). The contact angle measurements indicate that a surface with superior hydrophilicity was obtained for PES membranes. Two concentrations of PES (16 and 14.4 wt.%) and two different non-solvents (pure water and mixtures of water and IPA) were used for preparation of membranes. The morphological studies showed that the higher concentration of PES and the presence of IPA in the gelation media results in formation of a membrane with a dense top and sub-layer with small pores on the surface. The pure water flux of membranes was decreased when higher polymer concentration and mixtures of water and IPA were employed for membrane formation. On the other hand, the milk water permeation and protein rejection were increased using mixtures of water and IPA as non-solvent. Furthermore, the fouling analysis of the membranes demonstrated that the membrane surface with fewer tendencies for fouling was obtained.     

Preparation and characterization of crosslinked cellulose/sulfosuccinic acid membranes as proton conducting electrolytes

A series of crosslinked polymer electrolyte membranes were prepared by blending cellulose and sulfosuccinic acid (SA) for fuel cell applications. The crosslinking reaction of membranes occurred via the esterification between –OH of cellulose and –COOH of SA, as confirmed by FT-IR spectroscopy. Both the ion exchange capacity and the proton conductivity increased in proportion to the increase of SA concentrations due to the increasing portion of charged groups in the membrane. In contrast, the water uptake linearly increased up to 25 wt.% of SA concentration, above which it decreased abruptly. The maximum behavior of water uptake may be a result of competitive effect between the increasing number of ionic sites and the increasing degree of crosslinking with the SA concentrations. Wide angle X-ray scattering also showed that the crystalline structures of cellulose disappeared upon the introduction of SA. The mechanical properties of cellulose/SA membranes, i.e., tensile strength at break and Young’s modulus, showed a maximum at 15 wt.% of SA, as revealed by universal testing machine. These membranes exhibited good thermal stability up to 250 °C, as revealed by thermal gravimetric analysis.     

Water structure and selective permeation of cellulose-based membranes

The techniques used in the study of water in pores are extensively reviewed. Particular importance is given to ATR-FTIR, which allows the study of the active layer in the wet state. Taking further advantage of this technique, mathematical simulation studies on the ATR-FTIR spectra of water in cellulose acetate and cellulose acetate butyrate asymmetric membranes are performed. The OH-stretching band in the spectra of cellulose acetate membranes is deconvoluted into four components, which are ascribed to water clusters of different sizes. Results support the idea that the apparent rejection to salts may be associated to the size and number of water clusters whereas the flux depends on other factors, e.g. the thickness of the active layer.

The effect of salts, such as MgCl₂ and KCl, on the structure of water in the pores is also studied. The ATR-FTIR spectra of membranes previously equilibrated with solutions of such salts are deconvoluted.     

Wavelength-Selective Fluorescence of a Model Transmembrane Peptide: Constrained Dynamics of Interfacial Tryptophan Anchors

WALPs are prototypical, α-helical transmembrane peptides that represent a consensus sequence for transmembrane segments of integral membrane proteins and serve as excellent models for exploring peptide-lipid interactions and hydrophobic mismatch in membranes. Importantly, the WALP peptides are in direct contact with the lipids. They consist of a central stretch of alternating hydrophobic alanine and leucine residues capped at both ends by tryptophans. In this work, we employ wavelength-selective fluorescence approaches to explore the intrinsic fluorescence of tryptophan residues in WALP23 in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membranes. Our results show that the four tryptophan residues in WALP23 exhibit an average red edge excitation shift (REES) of 6 nm, implying their localization at the membrane interface, characterized by a restricted microenvironment. This result is supported by fluorescence anisotropy and lifetime measurements as a function of wavelength displayed by WALP23 tryptophans in POPC membranes. These results provide a new approach based on intrinsic fluorescence of interfacial tryptophans to address protein-lipid interaction and hydrophobic mismatch.     

蜂毒肽与多组分生物膜作用：电性与相态的影响

蜂毒肽非特异性地靶向杀伤细菌具有重要的生物医学应用前景. 利用荧光光谱与荧光显微考察了蜂毒肽与单组分、多组分磷脂膜的作用机制. 发现对于不同电性与相态的磷脂膜, 肽-膜作用呈现为稳定桶板型孔、非稳U型孔及变薄裂解等多种机制, 具有显著不同的内含物泄露效率. 多组分磷脂囊泡的泄露实验表明, 泄露由肽亲和性较强的磷脂组分决定. 相较于凝胶相磷脂, 蜂毒肽与液相磷脂的亲和性强, 凝胶-液相混合囊泡与纯液相磷脂囊泡的泄露性质相近; 相较于双电性磷脂, 蜂毒肽与负电性磷脂的亲和性强, 双电-负电混合囊泡与纯负电磷脂囊泡的泄露性质相近. 研究深化了多肽与多组分生物膜作用机制的理解.     

UV resonance Raman spectroscopy probes the localization of tryptophan‐containing antimicrobial peptides in lipid vesicles

In this work we employed UV resonance Raman spectroscopy with 229 nm excitation to study two tryptophan‐containing antimicrobial peptides with a broad‐spectrum activity against Gram‐positive and Gram‐negative bacteria: lactoferricin B (LfB, RRWQWRMKKLG) and pEM‐2 (KKWRWWLKALAKK). UV resonance Raman spectra of both peptides are dominated by tryptophan bands. Raman spectra of LfB and pEM‐2 in D₂O and 2,2,2‐trifluoro ethanol (TFE) have been measured and used to identify the hydrogen‐bond strength marker bands W6 and W17. The tryptophan doublet, W7, at 1340 and 1360 cm⁻¹ was used to detect an increase in the hydrophobicity of Trp environment in TFE. The spectra of LfB in complex with model cell membranes composed of zwitterionic dipalmitoylglycero‐phosphocholine (DPPC) or anionic dipalmitoyglycero‐phosphoglycerol (DPPG) lipid vesicles revealed a more hydrophobic Trp environment in DPPG, suggesting stronger interactions between the cationic peptide and anionic model cell membrane. Copyright © 2008 John Wiley & Sons, Ltd.     

Preparation of Cellulose Acetate Supported Zero-Valent Iron Nanoparticles for the Dechlorination of Trichloroethylene in Water

Chlorinated hydrocarbons are an immense concern for human health and the environment because they␣are highly toxic and are present in many contaminated sites. Zero-valent iron has been shown to be very effective for the dechlorination of chlorinated olefins and paraffins. This behavior is enhanced when the particle size is in the nanometer range. The activity of these nanoparticles is very high, and thus supporting the particles is important to preserve their chemical nature by inhibiting oxidation until they can be contacted with the chlorinated stream. In this paper, we present the preparation of membrane (cellulose acetate) supported zero-valent iron nanoparticles. The highly active nanoparticles were synthesized in a water-oil micro-emulsion, mixed with cellulose acetate-acetone solution, and then formed into a porous membrane by phase inversion. The unsupported iron particles and membrane supported iron particles were characterized using transmission electron microscopy. Batch experiments were conducted to characterize the activity of the supported zero-valent iron nanoparticles to dechlorinate trichloroethylene in water, as well as to investigate synergistic effects of the polymer support matrix.     

多纳米粒子穿越磷脂膜

本文采用耗散粒子动力学模拟方法研究了多纳米粒子与溶液中的磷脂膜相互作用. 模拟中选择纳米颗粒的形状分别为球状和柱状，并且在动力学过程中给它们设置了不同的初始速度. 根据纳米粒子穿膜在动力学过程中体现出不同的特性，分别定义了几种粒子穿越磷脂的模式，并且基于粒子之间的相互作用强度和粒子初始速度，描绘了穿膜模式的详细相图. 本文还进一步研究了体系能量、迴旋半径等参数在不同穿越模式中的动力学过程. 研究结果有助于人们理解纳米颗粒穿膜在生命活动过程中的作用.     

Sulfonated Aromatic Polymers and Organically Modified Montmorillonite Nanocomposite Membranes for Fuel Cells Applications

Aromatic polymers, such as sulfonated poly(2,6-dimethyl-1,4-phenylene oxide) (SPPO), sulfonated poly(ether ether ketone) (SPEEK), and sulfonated poly(ether sulfone) (SPES), at the optimum degrees of sulfonation (DS), are suggested and evaluated as alternatives to Nafion for direct methanol fuel cells (DMFCs) applications. To reduce the methanol cross-over, which decreases the efficiency of the cell, organically modified montmorillonite nanoclays (OMMT) were added at 1 wt% to the sulfonated matrices with the optimum DS. The X-ray diffraction (XRD) patterns of nanocomposite membranes proved that the nanoclay layers were exfoliated. The proton conductivity and methanol permeability of the membranes, as well as the ion-exchange capacity (IEC), were measured. The selectivity parameter, ratio of proton conductivity to methanol permeability, was identified at 25°C for the nanocomposite membranes and the results were compared with Nafion117. Finally, the DMFC performance tests were investigated at 70°C and 5 M methanol feed for the manufactured nanocomposite polyelectrolyte membranes (PEMs). The SPEEK-based nanocomposite membrane showed the highest maximum power density in comparison with Nafion 117 and SPES and SPPO nanocomposite membranes. The results indicated that the nanocomposite membranes were promising PEMs for DMFC applications.     

Ultrasound influence upon calcium carbonate precipitation on bacterial cellulose membranes

The effect of ultrasonic irradiation (40 kHz) on the calcium carbonate deposition on bacterial cellulose membranes was investigated using calcium chloride (CaCl₂) and sodium carbonate (Na₂CO₃) as starting reactants. The composite materials containing bacterial cellulose-calcium carbonate were characterized by means of scanning electron microscopy (SEM), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR) and color measurements. The polymorphs of calcium carbonate that were deposited on bacterial cellulose membranes in the presence or in the absence of ultrasonic irradiation were calcite and vaterite. The morphology of the obtained crystals was influenced by the concentration of starting solutions and by the presence of ultrasonic irradiation. In the presence of ultrasonic irradiation the obtained crystals were bigger and in a larger variety of shapes than in the absence of ultrasounds: from cubes of calcite to spherical and flower-like vaterite particles. Bacterial cellulose could be a good matrix for obtaining different types of calcium carbonate crystals.     

Permeability of cellulose acetate membranes to selected solutes

The permeability of cellulose 2.5-acetate films to several electrolytes and nonelectrolytes was measured. Permeabilities were determined by a desorption-rate method in which diffusion and distribution coefficients were measured. The rejection of the same solutes by modified cellulose acetate membranes in reverse osmosis experiments was also measured. A comparison was made between intrinsic water and solute permeabilities and the reverse osmosis semipermeability data using a noncoupled flow model of solute and water transport. The results were in reasonable agreement, indicating that flow coupling is not generally important in these films. Some of the factors, such as thermal history and casting environment, affecting the transport properties of acetone-cast cellulose acetate films were examined.     

Synthesis of carbon nanotubes directly over TEM grids aiming the study of nucleation and growth mechanisms

A novel approach to produce electron-transparent multi-layer membranes over TEM grids for transmission electron microscopy analysis is presented. The membranes have been used to grow and analyze carbon nanostructured materials in a thermal-chemical vapor deposition process using Ni and Cu as catalysts and silicon oxide thin films as support layers, at temperatures as high as 900 °C. It is important that conditions of the synthesis using the electron-transparent multi-layer membrane system are similar to those for a conventional chemical vapor deposition process, where oxidized silicon wafers are employed. In particular, the thickness of the silicon oxide and the catalyst layers are the same, and similar carbon tubular structures were grown using both substrates. The use of membranes was crucial especially for the study of the nucleation mechanism for carbon nanotubes. These electron-transparent multi-layer membranes are relatively easy to obtain and they can be used for transmission electron microscopy studies of high-temperature synthesis of different nanostructured materials.     

REDOR NMR on a hydrophobic peptide in oriented membranes

A method is presented for the calculation of REDOR dephasing for specifically labeled membrane-spanning peptides in uniformly aligned lipid bilayers under magic angle oriented sample spinning (MAOSS) conditions. Numerical simulations are performed for dephasing of (13)C signal by (15)N when the labels are placed in an alpha-helical peptide at the carbonyl of residue (i) and amide nitrogen of residue (i + 2) to show the dependency of REDOR echo intensity on the peptide tilt angle relative to the membrane normal. The approach was applied to the labeled transmembrane domain of phospholamban ([(15)N-Leu(37), (13)C-Leu(39)]PLBTM) incorporated into dimyristoylphosphatidylcholine bilayers. The dephasing observed for a random membrane dispersion showed that the peptide was alpha-helical in the region including the two labels, and dephasing in oriented membranes showed that the peptide helix was tilted by 25 degrees +/- 7 degrees relative to the bilayer normal. These results agree with those obtained by other spectroscopic methods.     

光谱分析法鉴别丙烯酸丁酯废水中的阴离子交换膜污染物

以回收实际丙烯酸丁酯废水有机酸的双极膜电渗析膜堆中的阴离子交换膜为研究对象,对膜使用前后的性能进行了表征,并综合通过衰减全反射傅里叶变换红外光谱(ATR-FTIR)、扫描电镜-能谱(SEM-EDS)和X射线光电子能谱(XPS)等手段对膜表面组分变化进行了表征。膜性能分析结果表明,使用后的阴离子交换膜受到污染,膜电阻增大,迁移数减小。能谱分析结果表明, 使用后阴离子交换膜表面污染物C和O元素含量(以原子百分比计)显著升高,分别从78.10%,8.34%升高到81.76%,12.05%, 表明膜污染物为含碳含氧物质。XPS谱图C1s峰的分析表明,-COO-Na+化学态的百分含量从污染前的8.5%升高到污染后的13.7%,表明膜污染物中含有-COO-Na+。ATR-FTIR分析结果表明,阴离子交换膜使用后,在1 561 cm-1处的吸收增强,而此波长正是-COO-M+(M为金属)的反对称伸缩振动峰,进一步验证了XPS分析结果。由于丙烯酸丁酯废水中含有聚丙烯酸钠,因此采用聚丙烯酸钠溶液污染阴离子交换膜,并对污染后离子交换膜进行膜性能、XPS和ATR-FTIR的分析。结果表明,聚丙烯酸钠导致阴离子交换膜膜电阻增大,迁移数减小,XPS谱图-COO-Na+化学态的百分含量增加,ATR-FTIR谱图1 561 cm-1处吸收增强。综合上述结果,聚丙烯酸钠是废水中造成阴离子交换膜污染的重要物质。光谱分析方法是离子交换膜污染层表征和污染物鉴别的有效手段。     

Membrane-water structural interactions in reverse osmosis transport

Reverse-osmotic water permeabilities, equilibrium water sorption levels, and rates of approach to sorption equilibrium were measured for a series of polymers, including hydroxyethyl methacrylate (HEMA), copolymers of HEMA and ethyl methacrylate (EMA), cellulose acetate, cellulose nitrate, and poly(urethans). Pronounced equilibrium solvent clustering behavior was observed for these systems as vapor saturation was approached in sorption experiments. However, clustering tendency was not found to be a function of total membrane water content at saturation but rather appears to be a function of the chemical nature of the polymer in question. Moreover, clustering of water molecules in (relatively) hydrophobic membranes resulted in low effective diffusivities (reverse osmotic permeability divided by equilibrium water content) whereas clustering in hydrophilic membranes led to higher effective water diffusivities. Clustering tendency was not as strong in the case of the weakly interacting membranes (i.e., the cellulose acetates). These conclusions were supported by theoretical diffusivity calculations. Predictions were based on analyses of transient sorption data, employing a dual-mode sorption model, and considering ordinary Fickian diffusion with simultaneous first-order reversible penetrant localization at water-binding sites in the polymer matrices. Means were found for correcting these diffusivity predictions to those values obtained experimentally under reverse osmosis conditions by accounting for the nonideality of the water flux under the latter conditions.