Effect of Phosphate on the Molecular Properties, Interactions, and Assembly of Engineered Spider Silk Proteins.

Phosphate plays a vital role in spider silk spinning and has been utilized in numerous artificial silk spinning attempts to replicate the remarkable mechanical properties of natural silk fiber. Its application in artificial processes has, however, yielded varying outcomes. It is thus necessary to investigate the origins and mechanisms behind these differences. By using recombinant silk protein SC-ADF3 derived from the garden spider Araneus diadematus, here, we describe its conformational changes under various conditions, elucidating the effect of phosphate on SC-ADF3 silk protein properties and interactions. Our results demonstrate that elevated phosphate levels induce the irreversible conformational conversion of SC-ADF3 from random coils to ß-sheet structures, leading to decreased protein solubility over time. Furthermore, exposure of SC-ADF3 to phosphate stiffens already formed structures and reduces the ability to form new interactions. Our findings offer insights into the underlying mechanism through which phosphate-induced ß-sheet structures in ADF3-related silk proteins impede fiber formation in the subsequent phases. From a broader perspective, our studies emphasize the significance of silk protein conformation for functional material formation, highlighting that the formation of ß-sheet structures at the initial stages of protein assembly will affect the outcome of material forming processes.

The adhesion capability of Staphylococcus aureus cells is heterogeneously distributed over the cell envelope.

Understanding and controlling microbial adhesion is a critical challenge in biomedical research, given the profound impact of bacterial infections on global health. Many facets of bacterial adhesion, including the distribution of adhesion forces across the cell wall, remain poorly understood. While a recent 'patchy colloid' model has shed light on adhesion in Gram-negative Escherichia coli cells, a corresponding model for Gram-positive cells has been elusive. In this study, we employ single cell force spectroscopy to investigate the adhesion force of Staphylococcus aureus. Normally, only one contact point of the entire bacterial surface is measured. However, by using a sine-shaped surface and recording force-distance curves along a path perpendicular to the rippled structures, we can characterize almost a hemisphere of one and the same bacterium. This unique approach allows us to study a greater number of contact points between the bacterium and the surface compared to conventional flat substrata. Distributed over the bacterial surface, we identify sites of higher and lower adhesion, which we call 'patchy adhesion', reminiscent of the patchy colloid model. The experimental results show that only some cells exhibit particularly strong adhesion at certain locations. To gain a better understanding of these locations, a geometric model of the bacterial cell surface was created. The experimental results were best reproduced by a model that features a few (5-6) particularly strong adhesion sites (diameter about 250 nm) that are widely distributed over the cell surface. Within the simulated patches, the number of molecules or their individual adhesive strength is increased. A more detailed comparison shows that simple geometric considerations for interacting molecules are not sufficient, but rather strong angle-dependent molecule-substratum interactions are required. We discuss the implications of our results for the development of new materials and the design and analysis of future studies.

Hydrophobin Bilayer as Water Impermeable Protein Membrane.

One of the most important properties of membranes is their permeability to water and other small molecules. A targeted change in permeability allows the passage of molecules to be controlled. Vesicles made of membranes with low water permeability are preferable for drug delivery, for example, because they are more stable and maintain the drug concentration inside. This study reports on the very low water permeability of pure protein membranes composed of a bilayer of the amphiphilic protein hydrophobin HFBI. Using a droplet interface bilayer setup, we demonstrate that HFBI bilayers are essentially impermeable to water. HFBI bilayers withstand far larger osmotic pressures than lipid membranes. Only by disturbing the packing of the proteins in the HFBI bilayer is a measurable water permeability induced. To investigate possible molecular mechanisms causing the near-zero permeability, we used all-atom molecular dynamics simulations of various HFBI bilayer models. The simulations suggest that the experimental HFBI bilayer permeability is compatible neither with a lateral honeycomb structure, as found for HFBI monolayers, nor with a residual oil layer within the bilayer or with a disordered lateral packing similar to the packing in lipid bilayers. These results suggest that the low permeabilities of HFBI and lipid bilayers rely on different mechanisms. With their extremely low but adaptable permeability and high stability, HFBI membranes could be used as an osmotic pressure-insensitive barrier in situations where lipid membranes fail such as desalination membranes.

Strong and Elastic Membranes via Hydrogen Bonding Directed Self-Assembly of Atomically Precise Nanoclusters.

2D nanomaterials have provided an extraordinary palette of mechanical, electrical, optical, and catalytic properties. Ultrathin 2D nanomaterials are classically produced via exfoliation, delamination, deposition, or advanced synthesis methods using a handful of starting materials. Thus, there is a need to explore more generic avenues to expand the feasibility to the next generation 2D materials beyond atomic and molecular-level covalent networks. In this context, self-assembly of atomically precise noble nanoclusters can, in principle, suggest modular approaches for new generation 2D materials, provided that the ligand engineering allows symmetry breaking and directional internanoparticle interactions. Here the self-assembly of silver nanoclusters (NCs) capped with p-mercaptobenzoic acid ligands (Na4 Ag44 -pMBA30 ) into large-area freestanding membranes by trapping the NCs in a transient solvent layer at air-solvent interfaces is demonstrated. The patchy distribution of ligand bundles facilitates symmetry breaking and preferential intralayer hydrogen bondings resulting in strong and elastic membranes. The membranes with Young's modulus of 14.5 ± 0.2 GPa can readily be transferred to different substrates. The assemblies allow detection of Raman active antibiotic molecules with high reproducibility without any need for substrate pretreatment.

Signatures of slip in dewetting polymer films.

Thin polymer films on hydrophobic substrates are susceptible to rupture and hole formation. This, in turn, initiates a complex dewetting process, which ultimately leads to characteristic droplet patterns. Experimental and theoretical studies suggest that the type of droplet pattern depends on the specific interfacial condition between the polymer and the substrate. Predicting the morphological evolution over long timescales and on the different length scales involved is a major computational challenge. In this study, a highly adaptive numerical scheme is presented, which allows for following the dewetting process deep into the nonlinear regime of the model equations and captures the complex dynamics, including the shedding of droplets. In addition, our numerical results predict the previously unknown shedding of satellite droplets during the destabilization of liquid ridges that form during the late stages of the dewetting process. While the formation of satellite droplets is well known in the context of elongating fluid filaments and jets, we show here that, for dewetting liquid ridges, this property can be dramatically altered by the interfacial condition between polymer and substrate, namely slip. This work shows how dissipative processes can be used to systematically tune the formation of patterns.

Using Knock-Out Mutants to Investigate the Adhesion of Staphylococcus aureus to Abiotic Surfaces.

The adhesion of Staphylococcus aureus to abiotic surfaces is crucial for establishing device-related infections. With a high number of single-cell force spectroscopy measurements with genetically modified S. aureus cells, this study provides insights into the adhesion process of the pathogen to abiotic surfaces of different wettability. Our results show that S. aureus utilizes different cell wall molecules and interaction mechanisms when binding to hydrophobic and hydrophilic surfaces. We found that covalently bound cell wall proteins strongly interact with hydrophobic substrates, while their contribution to the overall adhesion force is smaller on hydrophilic substrates. Teichoic acids promote adhesion to hydrophobic surfaces as well as to hydrophilic surfaces. This, however, is to a lesser extent. An interplay of electrostatic effects of charges and protein composition on bacterial surfaces is predominant on hydrophilic surfaces, while it is overshadowed on hydrophobic surfaces by the influence of the high number of binding proteins. Our results can help to design new models of bacterial adhesion and may be used to interpret the adhesion of other microorganisms with similar surface properties.

In-Depth Investigation of Copper Surface Chemistry Modification by Ultrashort Pulsed Direct Laser Interference Patterning.

Surface patterning in the micro- and nanometer-range by means of pulsed laser interference has repeatedly proven to be a versatile tool for surface functionalization. With these techniques, however, the surface is often changed not only in terms of morphology but also in terms of surface chemistry. In this study, we present an in-depth investigation of the chemical surface modification occurring during surface patterning of copper by ultrashort pulsed direct laser interference patterning (USP-DLIP). A multimethod approach of parallel analysis using visualizing, topography-sensitive, and spectroscopic techniques allowed a detailed quantification of surface morphology as well as composition and distribution of surface chemistry related to both processing and atmospheric aging. The investigations revealed a heterogeneous surface composition separated in peak and valley regions predominantly consisting of Cu2O, as well as superficial agglomerations of CuO and carbon species. The evaluation was supported by a modeling approach for the quantification of XPS results in relation to heterogeneous surface composition, which was observed by means of a combination of different spectroscopic techniques. The overall results provide a detailed understanding of the chemical and topographical surface modification during USP-DLIP, which allows a more targeted use of this technology for surface functionalization.

Dynamic Assembly of Class II Hydrophobins from T. reesei at the Air-Water Interface.

Class II hydrophobins are amphiphilic proteins produced by filamentous fungi. One of their typical features is the tendency to accumulate at the interface between an aqueous phase and a hydrophobic phase, such as the air-water interface. The kinetics of the interfacial self-assembly of wild-type hydrophobins HFBI and HFBII and some of their engineered variants at the air-water interface were measured by monitoring the accumulated mass at the interface via nondestructive ellipsometry measurements. The resulting mass vs time curves revealed unusual kinetics for a monolayer formation that did not follow a typical Langmuir-type of behavior but had a rather coverage-independent rate instead. Typically, the full surface coverage was obtained at masses corresponding to a monolayer. The formation of multilayers was not observed. Atomic force microscopy revealed formation and growth of non-fusing protein clusters at the interface. The mechanism of the adsorption was studied by varying the structure or charges of the protein or the ionic strength of the subphase, revealing that the lateral interactions between the hydrophobins play a role in their interfacial assembly. Additionally, a theoretical model was introduced to identify the underlying mechanism of the unconventional adsorption kinetics.

Binding Forces of Cellulose Binding Modules on Cellulosic Nanomaterials.

In this study, the interaction forces between different cellulosic nanomaterials and a protein domain belonging to cellulose binding modules family 1 (CBM1) were investigated at the molecular scale. Cellulose binding modules are protein domains found in carbohydrate active enzymes having an affinity toward cellulosic materials. Here, the binding force of a fusion protein containing a cellulose binding module (CBM1) produced recombinantly in E. coli was quantified on different cellulose nanocrystals immobilized on surfaces. Adhesion of the CBM on cellulose with different degrees of crystallinity as well as on chitin nanocrystals was examined. This study was carried out by single molecule force spectroscopy using an atomic force microscope, which enables the detection of binding force of individual molecules. The study contains a preliminary quantification of the interactions at the molecular level that sheds light on the development of new nanocellulose-based nanocomposites with improved strength and elasticity.

Slip-mediated dewetting of polymer microdroplets.

Classical hydrodynamic models predict that infinite work is required to move a three-phase contact line, defined here as the line where a liquid/vapor interface intersects a solid surface. Assuming a slip boundary condition, in which the liquid slides against the solid, such an unphysical prediction is avoided. In this article, we present the results of experiments in which a contact line moves and where slip is a dominating and controllable factor. Spherical cap-shaped polystyrene microdroplets, with nonequilibrium contact angle, are placed on solid self-assembled monolayer coatings from which they dewet. The relaxation is monitored using in situ atomic force microscopy. We find that slip has a strong influence on the droplet evolutions, both on the transient nonspherical shapes and contact line dynamics. The observations are in agreement with scaling analysis and boundary element numerical integration of the governing Stokes equations, including a Navier slip boundary condition.

Effect of Fluoride Treatment on the Acid Resistance of Hydroxyapatite.

The etching behavior of polycrystalline synthetic hydroxyapatite samples has been evaluated to explore the protective impact of fluoride on a tooth-like model system. Etching rates before and after fluoridation with a NaF solution at pH 6 were determined by atomic force microscopy. Despite a very low F concentration of ca. 0.2 atom % in the hydroxyapatite surface, a very strong effect on the acid resistance can be observed. Depending on the crystal orientation, etching in a NaAc buffer at pH 4.5 was completely inhibited for at least 5 min. The major part of the surface withstood etching even for more than 23 min. These results give new insights into how the amount of incorporated fluoride in hydroxyapatite correlates with its protective impact.

Adhesion Properties of Freestanding Hydrophobin Bilayers.

Hydrophobins are a family of small-sized proteins featuring a distinct hydrophobic patch on the protein's surface, rendering them amphiphilic. This particularity allows hydrophobins to self-assemble into monolayers at any hydrophilic/hydrophobic interface. Moreover, stable pure protein bilayers can be created from two interfacial hydrophobin monolayers by contacting either their hydrophobic or their hydrophilic sides. In this study, this is achieved via a microfluidic approach, in which also the bilayers' adhesion energy can be determined. This enables us to study the origin of the adhesion of hydrophobic and hydrophilic core bilayers made from the class II hydrophobins HFBI and HFBII. Using different fluid media in this setup and introducing genetically modified variants of the HFBI molecule, the different force contributions to the adhesion of the bilayer sheets are studied. It was found that in the hydrophilic contact situation, the adhesive interaction was higher than that in the hydrophobic contact situation and could be even enhanced by reducing the contributions of electrostatic interactions. This effect indicates that the van der Waals interaction is the dominant contribution that explains the stability of the observed bilayers.

Photoactivatable Mussel-Based Underwater Adhesive Proteins by an Expanded Genetic Code.

Marine mussels exhibit potent underwater adhesion abilities under hostile conditions by employing 3,4-dihydroxyphenylalanine (DOPA)-rich mussel adhesive proteins (MAPs). However, their recombinant production is a major biotechnological challenge. Herein, a novel strategy based on genetic code expansion has been developed by engineering efficient aminoacyl-transfer RNA synthetases (aaRSs) for the photocaged noncanonical amino acid ortho-nitrobenzyl DOPA (ONB-DOPA). The engineered ONB-DOPARS enables in vivo production of MAP type 5 site-specifically equipped with multiple instances of ONB-DOPA to yield photocaged, spatiotemporally controlled underwater adhesives. Upon exposure to UV light, these proteins feature elevated wet adhesion properties. This concept offers new perspectives for the production of recombinant bioadhesives.

Enhanced adhesion of Streptococcus mutans to hydroxyapatite after exposure to saliva.

Streptococcus mutans cells form robust biofilms on human teeth and are strongly related to caries incidents. Hence, understanding the adhesion of S. mutans in the human oral cavity is of major interest for preventive dentistry. In this study, we report on atomic force microscopy-based single-cell force spectroscopy measurements of S. mutans cells to hydroxyapatite surfaces. We observe for almost all measurements a significant difference in adhesion strength for S. mutans as well as for Staphylococcus carnosus cells. However, the increase in adhesion strength after saliva exposure is much higher for S. mutans cells compared to S. carnosus cells. Our results demonstrate that S. mutans cells are well adapted to their natural environment, the oral cavity. This ability promotes the biofilm-forming capability of that species and hence the production of caries-provoking acids. In consequence, understanding the fundamentals of this mechanism may pave a way towards more effective caries-reducing techniques.

The extracellular adherence protein (Eap) of Staphylococcus aureus acts as a proliferation and migration repressing factor that alters the cell morphology of keratinocytes.

Staphyloccocus aureus is a major human pathogen and a common cause for superficial and deep seated wound infections. The pathogen is equipped with a large arsenal of virulence factors, which facilitate attachment to various eukaryotic cell structures and modulate the host immune response. One of these factors is the extracellular adherence protein Eap, a member of the "secretable expanded repertoire adhesive molecules" (SERAM) protein family that possesses adhesive and immune modulatory properties. The secreted protein was previously shown to impair wound healing by interfering with host defense and neovascularization. However, its impact on keratinocyte proliferation and migration, two major steps in the re-epithelialization process of wounds, is not known. Here, we report that Eap affects the proliferation and migration capacities of keratinocytes by altering their morphology and adhesive properties. In particular, treatment of non-confluent HaCaT cell cultures with Eap resulted in cell morphology changes as well as a significant reduction in cell proliferation and migration. Eap-treated HaCaT cells changed their appearance from an oblong via a trapezoid to an astral-like shape, accompanied by decreases in cell volume and cell stiffness, and exhibited significantly increased cell adhesion. Eap had a similar influence on endothelial and cancer cells, indicative for a general effect of Eap on eukaryotic cell morphology and functions. Specifically, Eap was found to interfere with growth factor-stimulated activation of the mitogen-activated protein kinase (MAPK) pathway that is known to be responsible for cell shape modulation, induction of proliferation and migration of epithelial cells. Western blot analyses revealed that Eap blocked the phosphorylation of extracellular signal-regulated kinase 1 and 2 (Erk1/2) in keratinocyte growth factor (KGF)-stimulated HaCaT cells. Together, these data add another antagonistic mechanism of Eap in wound healing, whereby the bacterial protein interferes with keratinocyte migration and proliferation.

Nucleated dewetting in supported ultra-thin liquid films with hydrodynamic slip.

This study reveals the influence of the surface energy and solid/liquid boundary condition on the breakup mechanism of dewetting ultra-thin polymer films. Using silane self-assembled monolayers, SiO2 substrates are rendered hydrophobic and provide a strong slip rather than a no-slip solid/liquid boundary condition. On undergoing these changes, the thin-film breakup morphology changes dramatically - from a spinodal mechanism to a breakup which is governed by nucleation and growth. The experiments reveal a dependence of the hole density on film thickness and temperature. The combination of lowered surface energy and hydrodynamic slip brings the studied system closer to the conditions encountered in bursting unsupported films. As for unsupported polymer films, a critical nucleus size is inferred from a free energy model. This critical nucleus size is supported by the film breakup observed in the experiments using high speed in situ atomic force microscopy.

Influence of bidisperse self-assembled monolayer structure on the slip boundary condition of thin polymer films.

Alkylsilane self-assembled monolayers (SAMs) are often used as model substrates for their ease of preparation and hydrophobic properties. We have observed that these atomically smooth monolayers also provide a slip boundary condition for dewetting films composed of unentangled polymers. This slip length, an indirect measure of the friction between a given liquid and different solids, is switchable and can be increased [R. Fetzer et al., Phys. Rev. Lett. 95, 127801 (2005); O. Bäumchen et al., J. Phys.: Condens. Matter 24, 325102 (2012)] if the alkyl chain length is changed from 18 to 12 backbone carbons, for example. Typically, this change in boundary condition is affected in a quantized way, using one or the other alkyl chain length, thus obtaining one or the other slip length. Here, we present results in which this SAM structure is changed in a continuous way. We prepare bidisperse mixed SAMs of alkyl silanes, with the composition as a control parameter. We find that all the mixed SAMs investigated show an enhanced slip boundary condition as compared to the single-component SAMs. The slip boundary condition is accessed using optical and atomic force microscopy, and we describe these observations in the context of X-ray reflectivity measurements. The slip length, varying over nearly two orders of magnitude, of identical polymer melts on chemically similar SAMs is found to correlate with the density of exposed alkyl chains. Our results demonstrate the importance of a well characterized solid/liquid pair, down to the angstrom level, when discussing the friction between a liquid and a solid.

Intracellular Delivery of Poorly Soluble Polyphenols: Elucidating the Interplay of Self-Assembling Nanocarriers and Human Chondrocytes.

Increased molecular understanding of multifactorial diseases paves the way for novel therapeutic approaches requiring sophisticated carriers for intracellular delivery of actives. We designed and characterized self-assembling lipid-core nanocapsules for coencapsulation of two poorly soluble natural polyphenols curcumin and resveratrol. The polyphenols were identified as high-potential therapeutic candidates intervening in the intracellular inflammation cascade of chondrocytes during the progress of osteoarthritis. To elucidate the interplay between chondrocytes and nanocapsules and their therapeutic effect, we pursued a complementary analytical approach combining label-free visualization with biological assays. Primary human chondrocytes did not show any adverse effects upon nanocapsule application and coherent anti-Stokes Raman scattering images visualized their intracellular uptake. Further, by systematically blocking different uptake mechanisms, an energy independent uptake into the cells could be identified. Additionally, we tested the therapeutic effect of the polyphenol-loaded carriers on inflamed chondrocytes. Treatment with nanocapsules resulted in a major reduction of nitric oxide levels, a well-known apoptosis trigger during the course of osteoarthritis. For a more profound examination of this protective effect on joint cells, we pursued studies with atomic force microscopy investigations. Significant changes in the cell cytoskeleton as well as prominent dents in the cell membrane upon induced apoptosis were revealed. Interestingly, these effects could not be detected for chondrocytes which were pretreated with the nanocapsules. Overall, besides presenting a sophisticated carrier system for joint application, these results highlight the necessity of establishing combinatorial analytical approaches to elucidate cellular uptake, the interplay of codelivered drugs and their therapeutic effect on the subcellular level.

Characterization of alkylsilane self-assembled monolayers by molecular simulation.

Self-assembled monolayers (SAM) of dodecyltrichlorosilane (DTS) and octadecyltrichlorosilane (OTS) on silica are studied by molecular dynamics simulations at 298 K and 1 bar. The coverage (number of alkylsilane molecules per surface area) is systematically varied. The results yield insight into the properties of the alkylsilane SAMs, which complement experimental studies from the literature. Relationships are reported between thickness, tilt angle, and coverage of alkylsilane SAMs, which also hold for alkylsilanes other than DTS and OTS. They are interpreted based on the information on molecular ordering in the SAMs taken form the simulation data. System size and simulation time are much larger than in most former simulation works on the topic. This reduces the influence of the initial configuration as well as the periodic boundary conditions and hence minimizes the risk of artificial ordering. At the same time, more reliable statistics for the calculated properties can be provided. The evaluation of experimental data in the field is often based on strongly simplified models. The present simulation results suggest that some of these lead to errors, concerning the interpretation of experimental results, which could be avoided by introducing more realistic models.

Capillary droplet propulsion on a fibre.

A viscous liquid film coating a fibre becomes unstable and decays into droplets due to the Rayleigh-Plateau instability (RPI). Here, we report on the generation of uniform droplets on a hydrophobized fibre by taking advantage of this effect. In the late stages of liquid column breakup, a three-phase contact line can be formed at one side of the droplet by spontaneous rupture of the thinning film. The resulting capillary imbalance leads to droplet propulsion along the fibre. We study the dynamics and the dewetting speed of the droplet as a function of molecular weight as well as temperature and compare to a force balance model based on purely viscous dissipation.