Tubuliform silk protein: A protein with unique molecular characteristics and mechanical properties in the spider silk fibroin family

Orb–web weavers can produce up to six different types of silk and a glue for various functions. Tubuliform silk is unique among them due to its distinct amino acid composition, specific time of production, and atypical mechanical properties. To study the protein composing this silk, tubuliform gland cDNA libraries were constructed from three orb–weaving spiders Argiope aurantia, Araneus gemmoides, and Nephila clavipes. Amino acid composition comparison between the predicted tubuliform silk protein sequence (TuSp1) and the corresponding gland protein confirms that TuSp1 is the major component in tubuliform gland in three spiders. Sequence analysis suggests that TuSp1 shares no significant similarity with its paralogues, while it has conserved sequence motifs with the most primitive spider, Euagrus chisoseus silk protein. The presence of large side-chain amino acids in TuSp1 sequence is consistent with the frustrated β-sheet crystalline structure of tubuliform silk observed in transmission electron microscopy. Repeat unit comparison within species as well as among three spiders exhibits high sequence conservation. Parsimony analysis based on carboxy terminal sequence shows that Argiope and Araneus are more closely related than either is to Nephila which is consistent with phylogenetic analysis based on morphological evidence. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users. PACS 61.41; 87.14 Ee; 87.15 Cc     

Role of stochastic fluctuations in the charge on macroscopic particles in dusty plasmas

The currents which charge a macroscopic particle placed in a plasma consist of discrete charges; hence, the charge can undergo random fluctuations about its equilibrium value. These random fluctuations can be described by a simple model which, if the mechanisms for charging of macroscopic particles are known, makes it possible to determine the dependence of the temporal and amplitude characteristics of the fluctuations on the plasma parameters. This model can be used to study the effect of charge fluctuations on the dynamics of the macroscopic particles. The case of so-called plasma-dust crystals (i.e., highly ordered structures which develop because of strong interactions among macroscopic particles) in laboratory gaseous discharge plasmas is considered as an example. The molecular dynamics method shows that, under certain conditions, random fluctuations in the charge can effectively heat a system of macroscopic particles, thereby impeding the ordering process. Zh. éksp. Teor. Fiz. 115, 2067–2079 (June 1999)     

Role of stress on charge transfer through self-assembled alkanethiol monolayers on Au

We have studied charge transfer through alkanethiol molecules self-assembled on Au(111) substrates using interfacial force microscopy. Simultaneous measurement of the tip-substrate current and the normal interfacial force reveals the critical role of tip-film contact. Measurable currents are seen only for tip-applied stresses above about 20 MPa, after which the current rises exponential with stress. We suggest that charge transfer results from stress-induced band-gap states near the Fermi level in these normally highly insulating molecular films.     

Role of mesoscopic morphology in charge transport of doped polyaniline

In doped polyaniline (PANI), the charge transport properties are determined by mesoscopic morphology, which in turn is controlled by the molecular recognition interactions among polymer chain, dopant and solvent, Molecular recognition plays a significant role in chain conformation and charge delocalization. The resistivity of PANI doped by camphor sulfonic acid (CSA)/2-acrylo-amido-1-propane sulfonic acid (AMPSA)/dodecyl benzene sulfonic acid (DBSA) is around 0.02 Ω cm. PANI-CSA and PANI-AMPSA show a metallic positive temperature coefficient of resistivity above 150 K. with a finite value of conductivity at 1.4 K; whereas, PANI-DBSA shows hopping transport at low temperatures. The magnetoresistance is positive (negative) for PANI-CSA (PANI-AMPSA); and PANI-DBSA has a large positive MR. The behavior of MR suggests subtle variations in mesoscopic morphology between PANI-CSA and PANI-AMPSA.     

Light can transform the secondary structure of silk protein

Fibroin is the main component of silk and is expected to be used as a novel functional material in medicine and bioelectronics. The main secondary structures of this protein are of the random-coil and the β-sheet types. In this study, we carried out laser-induced transformation of the secondary structure, from the random-coil type to the β-sheettype, in solid fibroin films. We prepared two types of fibroin films with the random-coil structure. One is a fibroin film doped with a dye as a photosensitizer with a small amount (1 wt %), and the other is a neat fibroin film. The former was excited at 532 nm and the latter was excited at 266 nm. Irradiations were carried out with fluences much lower than each ablation threshold. The excitation of the dye at 532 nm did not affect the secondary structure of the random-coil type. By contrast, 266-nm laser irradiation, which excites tryptophan (an amino-acid residue) involved in fibroin, created the β-sheetdomain in the film. The structural transformation was revealed by infrared absorption spectroscopy and atomic force microscopy. Received: 1 August 2001 / Accepted: 2 August 2001 / Published online: 2 October 2001     

Broom-like and flower-like heterostructures of silver molybdate through pH controlled self assembly

Silver molybdate microrods are self-assembled into micron sized, broom-like and flower-like structures. Our investigations indicate that through a simple hydrothermal process, large scale production of such structure is possible. Using ammonium molybdate and silver nitrate solutions as precursors, we were able to show that the self assembled architectures were dependent on the pH of the starting precursor material. To understand the formation and destructions of the flower-like morphology, a systematic broad range (from acidic to basic) of pH-controlled experiments were performed and its influence on the structure/microstructure of synthesized materials was investigated. Scanning electron microscopy studies revealed that the morphology and microstructure of the products varied significantly by changing pH values from 3 to 8 during mixing of the reactants. pH = 3 and 4 resulted in the self assembly of monoclinic Ag₂(Mo₂O₇) microrods into broom-like structures, whereas pH = 5 resulted into the flower-like morphology of mixed phase of monoclinic and triclinic Ag₂Mo₂O₇. We also found that increasing the pH after a certain threshold value (for example pH > 6) resulted in total collapse of the flower-like morphology. Further increase of the pH to 7 and 8 resulted, the formation of microparticles of Ag₂MoO₄. A tentative scheme based on the pH-driven evolution of the self-assembly has been given to explain the formation of the observed heterostructures. Preliminary electrical characterization of thin films of the flower-like structures rendered non-linear current–voltage (I–V) responses. We also observed a strong hysteresis in the I–V responses of the flower-like structures developed under high bias conditions.     

Quantum size effect of surface-channeled charge carrier transport in Au nanoparticles-VO2 nanowire assembly

Confined charge carriers in nano-scaled structures have revealed great impacts on scientific and technological advances during the last few decades. Here we present quantum size effect of surface-channeled Au nanoparticles-VO₂ nanowire assembly fabricated via ac dielectrophoresis. Carrier injection is manipulated through Au nanoparticles decorated on the surface of single crystal VO₂ nanowire. Plateau structures are seen in the I–V characteristic of the assembly at 150 K, and the corresponding oscillation in channel conductance is analyzed in terms of quantum confinement induced two-dimensional layer of the carriers in a nanobelt formed around the insulating core of the VO₂ nanowire.     

Ultrasound-assisted air-jet spinning of silk fibroin-soy protein nanofiber composite biomaterials

Ultrasound utilizes a non-radiation technology that can meet modern standards to gain access to cheap, reliable and sustainable modern energy. Ultrasound technology can be implemented in the field of biomaterials for its exceptional potential in controlling the shape of nanomaterials. This study presents the first example of the production of soy and silk fibroin protein composite nanofibers in various ratios via combining ultrasonic technology with air-spray spinning. Characterization of ultrasonic spun nanofibers was performed by scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy (FTIR), X-ray powder diffraction (XRD), differential scanning calorimetry (DSC), thermogravimetric (TG) analysis, water contact angle, water retention, enzymatic degradation, and cytotoxicity assays. The effects that adjustments on the ultrasonic time have on the surface morphology, structures, thermal properties, hydrophilicity, water-uptake, bio-enzyme degradability, mechanical properties, and cytocompatibility of the material were examined. It was discovered that as the sonication time increased from 0 to 180 min, the beading phenomenon disappeared, forming nanofibers with uniform diameter and porosity; while the content of β-sheet crystals in the composites and their thermal stability gradually increased, the materials glass transition temperature decreased, and preferred mechanical properties were obtained. Additional studies show that the hydrophilicity, water retention capacity and enzymatic degradation rate were also enhanced by ultrasound, providing a favorable environment for cell attachment and proliferation. This study highlights the experimental and theoretical methods for ultrasound assisted air-jet spinning of biopolymer nanofibrous materials with tunable properties and high biocompatibility, which provide a wide range of applications in wound dressings and drug-carrying systems. This work shows great potential for a direct road to sustainable development of protein based fibers in the industry, thus promoting economic growth, and improving the health of the general population and well-being of wounded patients worldwide.     

Structure in protein solution changing the pH

Summary In this paper we present a small-angle neutron scattering measurement on concentrated aqueous solutions of lysozyme. The ranges for thepH and for the ionic strength of the solutions are chosen in order to match the physiological values at which the enzymatic activity of the protein is at his maximum. The net charge has been determined by separate tritation experiment. The form factor and the structure factor were extracted from the experimental data. The structure factor is quantitatively reproduced within a hard-sphere model with an attractive Yukawa-tail potential. The importance in this kind of systems of the attractive interaction, even at far from zero net charge is highlighted. Paper presented at the I International Conference on Scaling Concepts and Complex Fluids, Copanello, Italy, July 4–8, 1994.     

Ion specific protein assembly and hydrophobic surface forces

Large anions are attracted to hydrophobic surfaces while smaller, well solvated ions are repelled. Using a combination of explicit solvent and continuum model simulations we show that this leads to significant ion-specific protein-protein interactions due to hydrophobic patches on the protein surfaces. In solutions of NaI and NaCl we calculate the potentials of mean force and find that the resulting second virial coefficients for lysozyme correspond well with experiment. We argue that ionic interactions with nonpolar surface groups may play an important role for biomolecular assembly and Hofmeister-type effects.     

Electric potential decay on polyethylene: Role of atmospheric water on electric charge build-up and dissipation

Electrostatic potential decay on corona-charged low-density polyethylene (LDPE) was recorded as a function of position and time, using a macroscopic scanning electrode and also Kelvin force microscopy. Potential decays independently in adjacent sample areas until reaching equilibrium at negative values (4.6 ± 0.7 V), irrespective of the initial potential signal. Other observations already described in literature were confirmed: negative potential decays slower than positive potential and the relative humidity has a large effect on the dissipation rates. These results are discussed considering ion exchange associated to adsorption and desorption of water clusters at the solid–gas interface.     

Role of Interfacial Viscosity and pH in L-Phenylalanine,L-Tryptophan Molecular Rotors

Protein folding involves the aminoacid sequence to come forth and form an energy minimized structure. Recently molecular crowding leading to increase in viscosity is said to be one of the major concerns affecting protein folding. Many external fluorescent probes are used to detect such increases in viscosity. Since most of the protein sequences contain L-Phe and L-Trp, in this study we have used these aminoacids as probes to detect changes in viscosity. This study will help to advance the knowledge on molecular crowding effects in protein folding.     

The Role of Specific Side Groups and pH in Magnetization Transfer in Polymers

The nature of water–macromolecule interactions in aqueous model polymers has been investigated using quantitative measurements of magnetization transfer. Cross-linked polymer gels composed of 94% water, 3%N,N′-methylene-bis-acrylamide, and 3% functional monomer (acrylamide, methacrylamide, acrylic acid, methacrylic acid, 2-hydroxyethyl-acrylate, or 2-hydroxyethyl-methacrylate) were studied. Water–macromolecule interactions were modified by varying the pH and specific functional group on the monomer. The magnitudes of the interactions were quantified by measuring the rate of proton nuclear spin magnetization exchange between the polymer matrix and the water. This rate was highly sensitive to the presence of carboxyl side groups on the macromolecule. However, the dependence of the rate on pH was not consistent with simple acid/base-catalyzed chemical exchange, and instead, the data suggest that multiequilibria proton exchange, a wide distribution in surface group pKvalues, and/or a macromolecular structural dependence on pH may play a significant role in magnetization transfer in polymer systems. These model polymer gels afford useful insights into the relevance of chemical composition and chemical dynamics on relaxation in tissues.     

Preparation and mechanical properties of layers made of recombinant spider silk proteins and silk from silk worm

Layers of recombinant spider silks and native silks from silk worms were prepared by spin-coating and casting of various solutions. FT-IR spectra were recorded to investigate the influence of the different mechanical stress occurring during the preparation of the silk layers. The solubility of the recombinant spider silk proteins SO1-ELP, C16, AQ24NR3, and of the silk fibroin from Bombyx mori were investigated in hexafluorisopropanol, ionic liquids and concentrated salt solutions. The morphology and thickness of the layers were determined by Atomic Force Microscopy (AFM) or with a profilometer. The mechanical behaviour was investigated by acoustic impedance analysis by using a quartz crystal microbalance (QCMB) as well as by microindentation. The density of silk layers (d<300 nm) was determined based on AFM and QCMB measurements. At silk layers thicker than 300 nm significant changes of the half-band-half width can be correlated with increasing energy dissipation. Microhardness measurements demonstrate that recombinant spider silk and sericine-free Bombyx mori silk layers achieve higher elastic penetration modules E_EP and Martens hardness values H_M than those of polyethylenterephthalate (PET) and polyetherimide (PEI) foils.     

Dynamical measurements on viscoelastic behaviors of spiders in electro-dynamic loudspeakers

Spiders in electro-dynamic loudspeakers are most commonly concentrically corrugated fabric disks, and their viscoelastic behaviors affect the loudspeaker reproductions. A noncontact dynamic measuring technology is presented by a subwoofer closed box to excite the tested spiders pneumatically with a Laser Doppler Vibrometer (LDV) to measure the velocity of the moving spiders. Correlation techniques were employed to get an accurate and reliable acoustical transfer function between the measured velocity and sound pressure. The Young’s moduli of the tested spider composite materials were derived from the measured vibration modes. The creep effect and the level dependent behaviors of tested spiders were investigated. The results indicate that, the Young’s moduli of the tested spiders are frequency dependent. The mechanical stiffness increases with a small slope in low frequency range while a large slope in high frequency range. The loss factor exhibits the maximum around the resonance frequency, and after that it decreases with increasing frequency. The effective stiffness has a monotonic decrease with input voltage levels and the harder the spider, the less stiffness changes with input levels.     

Electrostatic assembly of protein lysozyme on DNA visualized by atomic force microscopy

In the present work, atomic force microscopy (AFM) has been used to study the assembly of protein lysozyme on DNA molecule. Based on the electrostatic interaction, the positively charged lysozyme can easily bind onto the negatively charged DNA molecule surface. The protein molecules appear as globular objects on the DNA scaffold, which are distinguishable in the AFM images. At the same time, lysozyme molecules can be assembled onto DNA as dense or sporadic pattern by varying the protein concentration. This work may provide fundamental aspects for building protein nanostructures and studying of DNA-protein interaction.     

Solution behavior of synthetic silk peptides and modified recombinant silk proteins

Spider dragline silk from Nephila clavipes possesses impressive mechanical properties derived in part from repetitive primary sequence containing polyalanine regions that self-assemble into crystalline β-sheets. In the present study, we have sought to understand more details of redox responses related to conformational transitions of modified silk peptides and a recombinant protein containing encoded methionine triggers. Regardless of the position of the methionine trigger relative to the polyalanine domain, chemical oxidation was rapid and slight increases in the α-helical structure and decreases in the β-sheet and random coil content were observed by CD and FTIR in the assembled silk-like peptides and the recombinant protein. CD results indicated that the decrease in β-sheet and random coil conformations, coupled with the increase in helical content during oxidation, occurred during the first 30 min of the reaction. No further conformational changes occurred after this time and the response was independent of methionine trigger location relative to the penta-alanine domain. These results were confirmed with fluorescence studies. The design, processing and utility of these modified redox triggered silk-like peptides and proteins suggest a range of potential utility, from biomaterials to engineered surface coatings with chemically alterable secondary structure and, thus, properties. PACS 87.14.Ee; 87.64.-t; 87.6.+2.     

Role of noncompensated space charge in propagation of ultrashort laser pulses in weakly ionized media

The problem of the propagation of a short pulse in fused silica has been considered in the vectorial approach at the free-electron plasma density below the breakdown threshold. For the first time, the noncompensated electric charge responsible for the considerable amplification of the longitudinal field component and superbroadening of the spectrum has been taken into account.     

Effects of pH and molecular charge on dipolar coupling interactions of solutes in skeletal muscle observed by DQF, 1H NMR spectroscopy

In this study we tested the effect of molecular charge and chirality as well as tissue pH on dipolar coupling interaction in skeletal muscle. These results were demonstrated by double quantum filtered, DQF, 1H NMR spectra acquired on permeable skeletal muscle samples dialyzed against buffered solutions containing three classes of solutes-electrolytes (lactate and Tris), zwitterions (alanine and glycine), and non-electrolytes (dioxane and ethanol)-as a function of pH ranging from 5.0 to 8.5. The results show that charge density on the protein filaments strongly influences dipolar coupling of solutes in muscle whereas charge on the solutes themselves has only a small effect. The frequency splitting of the dipolar coupled peaks for all the molecules tested was strongly affected by muscle pH. Higher pH increased negative charge density on the filaments and resulted in weaker dipolar coupling for anions and zwitterions but stronger coupling for the cation TRIS. Molecular charge per se or chirality did not affect the frequency splitting of the dipolar coupled peaks. The molecules, lactate, ethanol, and alanine, have scalar coupled spins and consequently a double quantum signal in solution. However, spectra acquired from these molecules in muscle showed an additional frequency splitting due to additional dipolar coupling interactions. Due to lack of scalar coupling, spectra from Tris, glycine, and dioxane showed no double quantum signal in solution but did when in muscle. All these observations can be explained by the fact that the net charge on protein filaments dominates the mechanism of dipolar coupling interactions in the highly anisotropic structures in muscle.