Article

Surface properties of spider silk particles in solution

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Abstract

Recombinant spider silk proteins, such as eADF4(C16), can be used for various applications. Colloidal particles of eADF4(C16) show potential as drug delivery systems. Tuning the colloidal properties of suspensions of eADF4(C16) particles represents a major prerequisite for their use in pharmaceutical formulations. In this study we determined the surface properties concerning inter-particle interactions by means of electrophoretic mobility and direct force measurements. The surface charge of eADF4(C16) spider silk particles was determined as a function of ionic strength and pH, respectively. The resulting electrophoretic mobility can be described using the O'Brien and White theory and is directly related to the amino acid sequence of the protein. We determined the extension of a fuzzy protein layer protruding into the solution by direct force measurements using a colloidal probe technique. This soft layer leads to deviations in the electrophoretic mobility and is responsible for additional repulsive forces at small separation distances. These steric forces lead to a stabilization of the particle suspension at high ionic strength.

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... Previously, it was shown that self-assembly of eADF4(C16) is triggered in the presence of phosphate ions (Pi). High concentrations of Pi (>400 mM) yield particles upon fast salting out and phase separation (Helfricht et al., 2013;Lammel et al., 2008;Slotta et al., 2008), while low Pi concentrations (<300 mM) trigger self-assembly into nano-fibrillar structures Slotta et al., 2007Slotta et al., , 2008 (Fig. 1 in Ref. (Humenik et al., 2015)). Assembly kinetics of eADF4(C16) fibrils could be explained by a nucleation dependent mechanism (Cohen et al., 2012;Eichner and Radford, 2011;Jarrett and Lansbury, 1993). ...
... In order to analyze the putative nucleation interface in more detail, b-sheet rich eADF4(C16) particles with a well-defined polymer-brush-like surface (Helfricht et al., 2013) were tested as seeds for fibril formation. eADF4(C16) was labeled either with the fluorophore N-hydroxysuccinimide-fluorescein or N-hydroxysuccinimide-rhodamine. ...
... eADF4(C16) monomers could also assemble on the surface of sub-micrometer eADF4(C16) particles (Fig. 6vi). Taking into account that eADF4(C16) particle surfaces reveal an interfacial layer of exposed brush-like protein chains (Helfricht et al., 2013), it is likely the sequence (polyalanine stretches) and its conformation on the interfacial layer surface that triggers fibril assembly on eADF4(C16) seeds and nuclei (represented as corresponding pale gray areas in Fig. 6). ...
Article
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Recombinant eADF4(C16) represents an engineered spider silk variant based on the sequence of the core domain of the natural dragline silk protein ADF4 of Araneus diadematus. Previously eADF4(C16) has been shown to self-assemble into cross- fibrils in a two-step process of nucleus formation and fibril growth. Here, it is shown that structurally converted low molecular weight oligomers can act as nuclei. Further, it could be determined that specifically potassium and phosphate ions strongly influence both nucleus formation as well as fibril growth. Nucleation of fibril assembly could be surpassed by seeding soluble protein with pre-assembled fibrils but also, unexpectedly, with eADF4(C16) sub-micrometer particles. The latter finding reveals that spider silk fibril assembly seems to be rather dependent on the protein sequence than on the structural features. Copyright © 2015. Published by Elsevier Inc.
... Electrophoretic methods are, in this respect, valuable techniques commonly used for the analytical characterization of spidroin particles. 7,13,14 A basic theory for electrophoresis of so-called hard particles, i.e., particles impermeable to ions and solvent, has been developed about a century ago and refined over the years to, e.g., account for surface ion-conduction processes and electric double layer polarization. 18 The numerical treatment of the standard electrokinetic model by O'Brien and White 19 is pivotal for analyzing situations where simplified analytical equations derived within the framework of the Debye−Huckel approximation are not applicable. ...
... 14,30,31 However, a recent study combining electrophoretic mobility with direct force measurements demonstrated that for such recombinant spidroin particles a pronounced diffuse interface with protruding protein segments exists, limiting the application of the classical O'Brien−White theory 19 (valid only for hard colloidal particles) to provide a comprehensive interpretation of the electrophoretic mobility as a function of pH and solution ionic strength. 13 Since the features of the spidroin particle surface depend critically on the production processing by salting-out, 32 a large number of parameters has to be taken into account. 12 Here, we address how far electrokinetic methods can be employed to characterize protein particles and to predict their overall colloidal stability. ...
... Beside both types of spidroin particles, silica particles (average diameter 6.8 μm, Bangs Laboratories Inc., Fishers, IN, U.S.A.) were immobilized on the functionalized dishes as "hard" internal standard. 13 The FluidFM setup enables the use of exchangeable colloidal probes. 35 First, the cantilever was approached near a silica particle, and an aspiration pressure of −800 mbar was applied. ...
Article
Colloidal particles have been prepared from polyanionic and polycationic recombinant spider silk protein. The amino acid sequences of these spider silk proteins are identical except for 16 residues bearing either a cationic or an anionic ionizable group. Electrophoretic titration showed that protonation of the acidic and basic amino acids had significant impact on the electrophoretic mobility of the protein particles and, in particular, on their point of zero mobility (PZM). The experimentally determined PZMs are in good agreement with the theoretical values evaluated on the basis of the relevant amino acid sequences. A comprehensive description of the electrokinetic properties of the recombinant spider silk protein particles as a function of pH and solution ionic strength was provided from adequate application of electrokinetic theory for soft particles. Within the framework of this formalism, spider silk protein particles are viewed as porous colloids penetrable for ions and characterized by a finite penetration length for the electroosmotic flow. The differentiated electrokinetic properties of the particles were shown to be solely governed by the electrohydrodynamic features of their poorly charged outer peripheral layer with a thickness of about 10-20 nm. This finding was further corroborated experimentally by demonstrating that electrokinetics of particles bearing an additional outer layer consisting of oppositely charged spider silk proteins is entirely dominated thereby. The presence of a fuzzy, ion-permeable particle interface with an extension of several tenths of a nanometer was confirmed by direct measurement of the resulting steric forces using the colloidal probe atomic force microscopy (AFM) technique.
... Furthermore, silk materials are nontoxic and have low immunogenicity [7,[98][99][100][101]. Finally, silk materials have excellent mechanical stability and a controllable format and size, and they can be stored in a dried state because of their reversible swelling behavior [102], offering unlimited opportunities for the fabrication, functionalization, and processing of robust biomaterials. Figure 1. ...
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For years, surgery, radiotherapy, and chemotherapy have been the gold standards to treat cancer, although continuing research has sought a more effective approach. While advances can be seen in the development of anticancer drugs, the tools that can improve their delivery remain a challenge. As anticancer drugs can affect the entire body, the control of their distribution is desirable to prevent systemic toxicity. The application of a suitable drug delivery platform may resolve this problem. Among other materials, silks offer many advantageous properties, including biodegradability, biocompatibility, and the possibility of obtaining a variety of morphological structures. These characteristics allow the exploration of silk for biomedical applications and as a platform for drug delivery. We have reviewed silk structures that can be used for local and systemic drug delivery for use in cancer therapy. After a short description of the most studied silks, we discuss the advantages of using silk for drug delivery. The tables summarize the descriptions of silk structures for the local and systemic transport of anticancer drugs. The most popular techniques for silk particle preparation are presented. Further prospects for using silk as a drug carrier are considered. The application of various silk biomaterials can improve cancer treatment by the controllable delivery of chemotherapeutics, immunotherapeutics, photosensitizers, hormones, nucleotherapeutics, targeted therapeutics (e.g., kinase inhibitors), and inorganic nanoparticles, among others.
... Measurement data were collected at the stationary planes for at least three times. The details on the procedure can be found elsewhere [57]. ...
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Clays are not only ubiquitous in nature, but they are also used in huge quantities in a broad range of industrial applications, such as thixotropic drilling fluids, ore pelletizers, waste disposal sealants, or fillers in polymer nanocomposites. In order to model environmental processes or to design new materials on a rational base, it is of prime importance to determine and possibly modify the interfacial properties of clay platelets at the solid/electrolyte interface. In this context, the fundamental question rises how far the stoichiometric interlayer charges as determined by the composition of the silicate layer correlates with the diffuse double-layer properties. Here, this question is addressed by means of a series of purposely synthesized sodium 2:1 layered silicates with defined composition and hence interlayer charge densities, respectively. Platelets of layered silicates of large enough diameter to perform AFM colloidal probe measurements were produced by melt synthesis. For comparison also, a natural muscovite mica has been included in this study. The diffuse layer properties in electrolyte solution have been determined by direct force measurements using the colloidal probe AFM technique and by electrokinetic measurements, respectively. We find that the diffuse layer potential decreases with increasing interlayer charge of the 2:1 layered silicates. This counterintuitive finding is attributed to ion adsorption and was further corroborated by determining the quantitative adsorption of polyelectrolytes, namely poly(amidoamine) dendrimers. Graphical abstract
... Spidroin particles substantially swell upon hydration (swelling factor of 2.3) inducing a drastic drop in elastic modulus from GPa in the dry state to MPa in the wet state [145], depending on the molecular weight of the respective spidroins. Direct force measurements using a colloidal probe technique revealed a polymer brush-like surface i.e., an ion-permeable particle interface protruding several tenths of nm into the solution [146,147]. Interestingly, it has been shown that particle surfaces can seed fibril growth (Fig. 6.4d) [102]. ...
Chapter
The extraordinary mechanical properties of spider silk fibers result from the interplay of composition, structure and self-assembly of spider silk proteins (spidroins). Genetic approaches enabled the biotechnological production of recombinant spidroins which have been employed to unravel the self-assembly and spinning process. Various processing conditions allowed to explore non-natural morphologies including nanofibrils, particles, capsules, hydrogels, films or foams. Recombinant spider silk proteins and materials made thereof can be utilized for biomedical applications, such as drug delivery, tissue engineering or 3D-biomanufacturing.
... Using ionic liquids instead of aqueous buffers and high potassium phosphate concentrations to induce phase separation and nucleation in the protein solution or ultrasonication for particle production allowed enhanced size control and a reduced polydispersity index (Elsner et al. 2015;Lucke et al. 2015). eADF4(C16) (engineered Araneus diadematus fibroin 4) particles show a brush-like outer layer with protruding protein strands and a thickness of 30-50 nm covering a solid inner core (Helfricht et al. 2013). Importantly, no posttreatment with dehydrating agents is necessary to obtain water-insoluble particles, since the β-sheet content is high after the salting-out process . ...
Article
Spider silk fibers have a sophisticated hierarchical structure composed of proteins with highly repetitive sequences. Their extraordinary mechanical properties, defined by a unique combination of strength and extensibility, are superior to most man-made fibers. Therefore, spider silk has fascinated mankind for thousands of years. However, due to their aggressive territorial behavior, farming of spiders is not feasible on a large scale. For this reason, biotechnological approaches were recently developed for the production of recombinant spider silk proteins. These recombinant proteins can be assembled into a variety of morphologies with a great range of properties for technical and medical applications. Here, the different approaches of biotechnological production and the advances in material processing toward various applications will be reviewed.
... Measurement data were collected at the stationary planes for at least three times. The details on the procedure can be found elsewhere [57]. ...
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The adhesion between colloidal silica particles and modified electrodes has been studied by direct force measurements with the colloidal probe technique based on the atomic force microscope (AFM). The combination of potentiostatic control of gold electrodes and their chemical surface modification by self-assembled monolayers (SAMs) allows for the decoupling of forces due to the electrical double layers and chemical functionality of the solid/liquid interface. Adhesion on such electrodes can be tuned over a large range in dependence of the externally applied potential and the aqueous solution's ionic strength. By utilizing cantilevers with a high force constant, it is possible to separate the various contributions to the adhesion in an unambiguous manner. These contributions comprise diffuse layer overlap, van-der Waals forces, solvent exclusion, and electrocapillarity. A quantitative description of the observed adhesion forces is obtained by taking into account the surface roughness of the silica particle. The main component in the adhesion, which is tuned by the external potential, originates from the overlap of the electrical double layers. Instead, effects due to electrocapillarity are only of minor importance. Based on our quantitative analysis a new approach is proposed that allows tuning the adhesion force as a function of the externally applied potential. This approach is supposed to have important implications for the design of microelectromechanical systems (MEMS), the development of electrochemical sensors as well as for micro- and nanomanipulation.
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The interaction energy between microscopic bodies is almost exclusively determined assuming perfectly smooth and geometrically regular surfaces. Quite often, such interactions fail to explain several colloidal phenomena. These inexplicable behaviors of colloidal systems are generally ascribed to surface chemical and morphological heterogeneities. Here, we employ the surface element integration technique to determine the interaction energy between surfaces containing morphological heterogeneity. Random asperities are generated to represent surface morphological heterogeneity (roughness), and their influence on the DLVO interaction potential is investigated. Incorporation of surface roughness causes a significant reduction in the repulsive interaction energy, the extent of which depends on the size of the asperities and their densities on the surface. Predictions of interaction energy indicate that the DLVO interaction energy profiles for rough surfaces deviate significantly from those derived assuming perfectly smooth surfaces, particularly at very short separation distances.
Article
An atomic force microscope has been used to study the forces between a silica sphere in the colloidal size range and silica or mica flat surfaces as a function of distance of separation. At low ionic strength, independent electrokinetic measurements (ζ potentials) of both the spheres (by electrophoresis) and flat surfaces (by streaming potential) under the same conditions show excellent agreement with the diffuse double layer potentials derived from the force data using conventional DLVO theory. At higher ionic strength, the electrokinetically derived potentials were found to deviate from those derived from the fitted atomic force microscopy data, and a short range steric type repulsion was observed between the surfaces, the magnitude of which increased with decreasing pH.
Article
The forces between hydrophilic and hydrophobic silica particles and an air bubble were measured in pure water and in NaCl solutions using an atomic force microscope. In addition to the expected double-layer and van der Waals forces, strong long-range attractive forces were also observed. A long-range attraction was also measured between a hydrophilic silica particle and a hydrophobic silica plate. A gas bubble thus behaves like a hydrophobic surface. The long-ranged attractive component of the force disappeared when the anionic surfactant sodium dodecylsulfate (SDS) was added to the solution. This effect is explicable in terms of surfactant adsorption at the hydrophobic interfaces which renders them hydrophilic. A ''thermodynamic'' model is proposed that appears to be consistent with these and previous force and wetting experiments on hydrophobic surfaces. It is also demonstrated that a nonzero water contact angle on clean hydrophilic silica and similar hydrophilic surfaces can arise from DLVO forces alone and is not necessarily an indication of surface contamination or some hydrophobic component in the force.
Article
A general expression is derived for the electrophoretic mobility of a soft particle, i.e., a spherical hard colloidal particle of radius a coated with a layer of polyelectrolytes of thickness d in an electrolyte solution, In the limit of d → 0, the mobility expression tends to that for a spherical hard particle (Ohshima et al., J. Chem. Soc. Faraday Trans.2 79, 1613 (1983)), whereas in the limit of a → ∞, it tends to that for a plate-like soft particle derived previously (J. Colloid Interface Sci.130, 281 (1989)). Also in the limit of a → 0 and low potentials, the obtained mobility expression tends to a mobility formula for a spherical polyelectrolyte (a charged porous sphere with no particle core) derived by Hermans and Fujita (K. Ned. Akad. Wet. Proc. Ser. B58, 182 (1955)). A simple approximate analytic mobility expression is presented.
Article
Electrophoretic mobilities (EPM) of negatively charged latex spheres were measured as a function of salt type and salt concentration. The measured values of EPM were analyzed using a standard electrokinetic model that includes double layer relaxation and the Poisson–Boltzmann model of diffuse double layer. Calculated values of EPM were in good agreement with experimental data taken in simple 1:1 (KCl) and 1:2 (Na2SO4) electrolyte solutions without using any fit parameters. For 2:1 electrolytes (CaCl2 and MgCl2), however, the magnitude of EPM calculated by the model was higher than the measured values of EPM at higher electrolyte concentrations. The difference between measured and calculated EPM was reduced by assuming the distance of slipping plane x s = 0.25nm or by assuming the decrease of the magnitude of surface charge density from −0.07 to −0.025C/m2. These are probably due to the accumulation of divalent counterions in the vicinity of a particle’s surface.
Article
Polystyrene microspheres having roughly the same size but different negative surface charge densities were prepared by emulsion polymerization. The amount of sulfate groups on the surface of the particles was controlled by variation of the amount and the decomposition rate of the initiator used, potassium, persulfate. After the cleaning process involving dialysis and extensive ultrafiltration the surface-charge density of the samples was determined and their electrokinetic behavior was studied. A simple model based on the Gouy-Chapman theory and the O’Brian-White approach allows the calculation of the dependence of the electrophoretic mobility on salt concentration. Comparison of the theoretical and experimental curves showed that they were in good agreement in a number of qualitative features. Moreover, the model revealed that a monotonously increasing zeta potential with falling electrolyte concentration results in a mobility maximum, and that this so-called atypical behavior is in accordance with the standard electrokinetic theory. No ion adsorption mechanism or the existence of a charged hairy layer, current standard explanations for this anomality, had to be invoked.
Article
Electrophoretic mobility studies of highly charged amidine latex particles were carried out to validate the standard electrokinetic model. The electrophoretic mobilities were measured on a standard commercial laser Doppler velocimetry electrophoresis apparatus. In a plot against the salt concentration, a pronounced mobility maximum was observed. This observation, which is a direct consequence of the standard electrokinetic model, showed good agreement with theory.
Article
The engineered and recombinant spider silk protein eADF4(C16) has been shown to be a promising biomaterial for the use as drug delivery system. In previous studies, eADF4(C16) particles were loaded with low molecular weight drugs exhibiting a positive net-charge and sufficient hydrophobicity. Here, we demonstrate that also macromolecular drugs like proteins can be loaded on eADF4(C16) particles. Using lysozyme as a model protein, remarkably high loading of up to 30% [w/w] was feasible and high loading efficiencies of almost 100% were obtained. Furthermore, using confocal laser scanning microscopy, it is demonstrated that fluorescently labeled lysozyme is not only adsorbed to the negatively charged particles' surface, but also diffusing into the matrix of eADF4(C16) particles. The release of lysozyme is shown to be dependent on the ionic strength and pH of the release medium. To improve the long-term stability of eADF4(C16) containing dispersions, lyophilization is shown as a suitable tool. Disaccharides (sucrose, trehalose) and mannitol served as stabilizers to prevent aggregation and/or particle degradation during freeze-drying. The slowly biodegradable eADF4(C16) particles are a promising new particulate drug carrier system for the delivery of susceptible drugs like therapeutic proteins.
Article
Spider silk has been in the focus of research mainly due to the superior mechanical characteristics of silk fibers. However, it has been previously shown that spider silk proteins can also adopt other morphologies such as submicroparticles. This study examines the applicability of such particles as drug carriers. Particle characterization revealed that particles made of the engineered spider silk protein eADF4(C16) are colloidally stable in solution. Here, it is shown that small molecules with positive net-charge can diffuse into the negatively charged spider silk protein matrix driven by electrostatic interactions. The loading efficiencies correlate with the distribution coefficient (logD) of small molecules of weak alkaline nature. Interestingly, constant release rates can be realized for a period of two weeks at physiological conditions in vitro, with accelerated release rates within acidic environments. Enzymatic degradation studies of eADF4(C16) particles indicated that the silk proteins degrade slowly and the particles decrease in size. Along with their all-aqueous and easy preparation, drug loaded eADF4(C16) particles provide a high potential for diverse applications in which controlled release from biodegradable carriers is desired.
Article
Steric forces between polymer brushes and atomic force microscope tips were investigated. We studied two systems: polystyrene(PS) grafted to silicon in cyclohexane and poly(ethylene oxide)/poly(methacrylic acid) (PEO/PMAA) diblock copolymer adsorbed with the PMAA block to aluminum oxide in aqueous medium. On approach exponentially decaying repulsive forces were observed in both systems. With a homemade heat stage we could adjust the temperature. Increasing the temperature between 19 and 53 degrees C led to a linear increase of the decay length for PS in cyclohexane. Also the work required to bring the tip to a certain distance increased roughly linearly with temperature. This supports the view that the repulsion is of entropic origin. At the same time this demonstrates that the temperature dependence of surface forces could be routinely measured. For PEO in water the repulsive force was not significantly affected by a change in temperature. Approaching and retracting parts of force curves measured with PS in cyclohexane were in most cases indistinguishable. In contrast, for PEO in water a significant hysteresis was observed. This might be caused by an escape of polymers underneath the tip of the atomic force microscope. When retracting the tip in some cases the stretching of individual polymers was observed in both systems. Stretching force vs distance curves could be described by a wormlike chain model with typical persistence lengths of 1 nm.
Article
Due to their extraordinary mechanical and biochemical properties, silks have long been in focus of research. In vivo, fibers are formed from silk proteins, in vitro, however, a variety of materials can be produced in addition to fibers including capsules, particles, films, foams, and gels. The versatility of silk proteins, along with their biocompatibility, biodegradability, and potential for processing in aqueous solution under ambient conditions make silk-based materials good candidates for biomedical applications such as drug delivery systems and scaffolds for tissue engineering. Here, we summarize recent progress in research employing recombinantly produced engineered spider silk proteins with a focus on the fundamentals of silk protein processing. We highlight recombinant spider silk films and particles as morphologies that represent model systems with adjustable material properties controlled by process parameters.
Article
Interaction forces between ionizable surfaces across an electrolyte solution on the Poisson-Boltzmann level are discussed within the constant regulation approximation. The chemical response of each surface is expressed in terms of two parameters, namely, the diffuse layer potential and the regulation parameter p. Both parameters are easily available because they arise naturally within classical equilibrium models for a single noninteracting surface. This approximation, thus, eliminates the need to treat the more intricate problem of two chemical adsorption equilibria coupled to the overlapping double layers between the surfaces. The ensuing simplicity makes this approach extremely versatile for the analysis of experimental data. The classical boundary condition of constant potential corresponds to p = 0, and that of constant charge corresponds to p = 1. While this approximation is rigorously correct at large separations, we find that it remains excellent down to contact in many realistic situations, such as in symmetric or asymmetric systems involving metal oxides or silica described by the 1-pK basic Stern model.
Article
Biomaterials, having evolved over millions of years, often exceed man-made materials in their properties. Spider silk is one outstanding fibrous biomaterial which consists almost entirely of large proteins. Silk fibers have tensile strengths comparable to steel and some silks are nearly as elastic as rubber on a weight to weight basis. In combining these two properties, silks reveal a toughness that is two to three times that of synthetic fibers like Nylon or Kevlar. Spider silk is also antimicrobial, hypoallergenic and completely biodegradable. This article focuses on the structure-function relationship of the characterized highly repetitive spider silk spidroins and their conformational conversion from solution into fibers. Such knowedge is of crucial importance to understanding the intrinsic properties of spider silk and to get insight into the sophisticated assembly processes of silk proteins. This review further outlines recent progress in recombinant production of spider silk proteins and their assembly into distinct polymer materials as a basis for novel products.
Article
The van der Waals and electrostatic interaction energies between a single particle and a flat plate were measured using the optical technique of total internal reflection microscopy (TIRM). The particles used were 15-µm-diameter polystyrene latex spheres and the flat plate was a polished BK-7 glass slide. The measurements were performed in aqueous solutions of ionic strength ranging from 3 to 7 mM, and the particle-plate separation distances ranged from approximately 25 to 300 nm. During measurement, the particle was trapped in a secondary energy well formed by the repulsive electrostatic and attractive van der Waals forces; however, the particle was free to undergo Brownian motion at all times. These measurements, which capitalize on the extreme sensitivity of the TIRM technique, are believed to be the first direct measurement of the van der Waals interaction energy in aqueous solutions at separation distances where retardation effects are substantial. Comparison of the measured energy wells with predictions made with traditional energy equations produced only fair agreement; specifically, the measured well depths were consistently lower than predicted. However, when the measured results were compared with predictions made using the recent model of L. Suresh and J. Y. Walz ([J. Colloid Interface Sci. 183, 199 (1996)] for rough surfaces, very good agreement was obtained. The asperity heights yielding the best agreement ranged between 14 and 33 nm, with an average height of 26 nm. This value is consistent with previous estimates of the roughness height obtained by measuring the particle sedimentation velocity [J. Y. Walz and L. Suresh, J. Chem. Phys. 103, 10714 (1995)]. Copyright 1997 Academic Press.
Article
Spider silk proteins have mainly been investigated with regard to their contribution to mechanical properties of the silk thread. However, little is known about the molecular mechanisms of silk assembly. As a first step toward characterizing this process, we aimed to identify primary structure elements of the garden spider's (Araneus diadematus) major dragline silk proteins ADF-3 and ADF-4 that determine protein solubility. In addition, we investigated the influence of conditions involved in mediating natural thread assembly on protein aggregation. Genes encoding spider silk-like proteins were generated using a cloning strategy, which is based on a combination of synthetic DNA modules and PCR-amplified authentic gene sequences. Comparing secondary structure, solubility, and aggregation properties of the synthesized proteins revealed that single primary structure elements have diverse influences on protein characteristics. Repetitive regions representing the largest part of dragline silk proteins determined the solubility of the synthetic proteins, which differed greatly between constructs derived from ADF-3 and ADF-4. Factors, such as acidification and increases in phosphate concentration, which promote silk assembly in vivo generally decreased silk protein solubility in vitro. Strikingly, this effect was pronounced in engineered proteins comprising the carboxyl-terminal nonrepetitive regions of ADF-3 or ADF-4, indicating that these regions might play an important role in initiating assembly of spider silk proteins.
Article
The current theoretical approaches to electrokinetics of gels or polyelectrolyte layers are based on the assumption that the position of the very interface between the aqueous medium and the gel phase is well defined. Within this assumption, spatial profiles for the volume fraction of polymer segments (phi), the density of fixed charges in the porous layer (rho fix), and the coefficient modeling the friction to hydrodynamic flow (k) follow a step-function. In reality, the "fuzzy" nature of the charged soft layer is intrinsically incompatible with the concept of a sharp interface and therefore necessarily calls for more detailed spatial representations for phi, rho fix, and k. In this paper, the notion of diffuse interface is introduced. For the sake of illustration, linear spatial distributions for phi and rho fix are considered in the interfacial zone between the bulk of the porous charged layer and the bulk electrolyte solution. The corresponding distribution for k is inferred from the Brinkman equation, which for low phi reduces to Stokes' equation. Linear electrostatics, hydrodynamics, and electroosmosis issues are analytically solved within the context of streaming current and streaming potential of charged surface layers in a thin-layer cell. The hydrodynamic analysis clearly demonstrates the physical incorrectness of the concept of a discrete slip plane for diffuse interfaces. For moderate to low electrolyte concentrations and nanoscale spatial transition of phi from zero (bulk electrolyte) to phi o (bulk gel), the electrokinetic properties of the soft layer as predicted by the theory considerably deviate from those calculated on the basis of the discontinuous approximation by Ohshima.
Article
Colloidal silica is known to be stable at high salt concentrations and low pH, where silica is basically uncharged. This observation is in qualitative disagreement with the theory of Derjaguin, Landau, Verwey, and Overbeek (DLVO), which predicts rapid aggregation (or coagulation) under these conditions. This study reports a very different behaviour for Stöber-type silica heated at 800 degrees C, as these particles follow DLVO theory quantitatively. Unheated samples behave approximatively according to DLVO theory, but they show systematic deviations, in particular, featuring higher stability at low pH. The heat treatment also substantially modifies the charging properties, as heated particles show titratable surface charge densities in the range expected for the water-silica interface, while much higher charge densities are observed for the unheated samples. The electrophoretic mobilities, on the other hand, are hardly influenced by the heat treatment. We suspect that the suspension stability of the unheated particles is influenced by the presence of a hairy-layer of polysilicilic acid chains on the surface.
Article
In this report, the status quo and recent progress in electrokinetics are reviewed. Practical rules are recommended for performing electrokinetic measurements and interpreting their results in terms of well-defined quantities, the most familiar being the zeta-potential or electrokinetic potential. This potential is a property of charged interfaces and it should be independent of the technique used for its determination. However, often the zeta-potential is not the only property electrokinetically characterizing the electrical state of the interfacial region; the excess conductivity of the stagnant layer is an additional parameter. The requirement to obtain the zeta-potential is that electrokinetic theories be correctly used and applied within their range of validity. Basic theories and their application ranges are discussed. A thorough description of the main electrokinetic methods is given; special attention is paid to their ranges of applicability as well as to the validity of the underlying theoretical models. Electrokinetic consistency tests are proposed in order to assess the validity of the zeta-potentials obtained. The recommendations given in the report apply mainly to smooth and homogeneous solid particles and plugs in aqueous systems; some attention is paid to nonaqueous media and less ideal surfaces.
Article
The electrostatic, hydrodynamic, and swelling properties of a well-defined, functionalized carboxymethyldextran (CMD) polysaccharide are investigated in aqueous NaNO3 solution over a broad ionic strength range. The impact of the polycarboxylate charge and molar mass of the CMD macromolecules on their electrohydrodynamic features is thoroughly examined by combined protolytic titration, dynamic light scattering, and electrokinetic analyses. Electrophoretic mobility data obtained for sufficiently high electrolyte concentrations reveal a typical soft particle behavior. Upon decrease of the ionic strength, mobilities strongly increase in magnitude while significant electrostatic swelling takes place, as reflected in a decrease in the diffusion coefficients. CMD entities undergo conformational transitions from compact random coil at large ionic strengths to swollen coil and possibly a wormlike structure at lower NaNO3 concentrations. The magnitude of the variations in size and mobility with electrolyte concentration strongly depends on the overall charge of the CMD entity as well as on its molar mass. These factors control the stiffness of the constituent polymer chains and thus the degree of macromolecular permeability ("softness"). Using the soft-diffuse interface formalism previously developed for the electrohydrodynamics of charged permeable macromolecules, a quantitative analysis of the electrophoretic mobility data is presented. The measured values of the diffusion coefficient and space charge density Gamma degrees, as evaluated independently from the modeling of potentiometric titration curves, are taken into account in a self-consistent manner. It is found that large CMD entities of low charge densities are the most permeable to flow penetration with a limited heterogeneous electrostatic stiffening of the chains, whereas small CMD entities of larger Gamma degrees significantly expand upon lowering the ionic strength, giving rise to a strong anisotropy for the spatial distribution of polymer chain density.