The epidemic spread of many viral infections is mediated by the environmental conditions and influenced by the ambient humidity. Single virus particles have been mainly visualized by atomic force microscopy (AFM) in liquid conditions, where the effect of the relative humidity on virus topography and surface cannot be systematically assessed. In this work, we employed multi-frequency AFM, simultaneously with standard topography imaging, to study the nanoscale wetting of individual Tobacco Mosaic virions (TMV) from ambient relative humidity to water condensation (RH > 100%). We recorded amplitude and phase vs. distance curves (APD curves) on top of single virions at various RH and converted them into force vs. distance curves. The high sensitivity of multifrequency AFM to visualize condensed water and sub-micrometer droplets, filling gaps between individual TMV particles at RH > 100%, is demonstrated. Dynamic force spectroscopy allows detecting a thin water layer of thickness ~1 nm, adsorbed on the outer surface of single TMV particles at RH < 60%.

A probabilistic discrete model for 2D protein crystal growth is presented. This model takesinto account the available space and can describe growing processes of a different nature due to theversatility of its parameters, which gives the model great flexibility. The accuracy of the simulation istested against a real recrystallization experiment, carried out with the bacterial protein SbpA fromLysinibacillus sphaericus CCM2177, showing high agreement between the proposed model and theactual images of the crystal growth. Finally, it is also discussed how the regularity of the interface(i.e., the curve that separates the crystal from the substrate) affects the evolution of the simulation.

We investigate the electrospinning of small molecules, specifically designed peptide derivatives of the pyrazole-isothiazole scaffold. Such non-natural peptides enhance the spectrum of fundamental materials used for electrospinning. Unlike standard electrospun materials, our peptides are not polymeric, but able to aggregate in solution and especially during processing. They contain donor/acceptor groups that can form hydrogen bonds, and groups that are able to generate π-stacking interactions, which are known as important requirements for assembly processes. The pyrazole-isothiazole derivatives were synthesized by means of a 1,3-dipolar cycloaddition reaction, which is completely regioselective, affording only one isomer. We demonstrate that our compounds can be electrospun from fluoroalcohol solution into solid, quasi-endless micro- and nanofibers. The electrospinnability varies substantially, depending on the amino acids linked to the scaffold. Some compounds provide only short fibers, while Fmoc-glycyl-(N-benzyl)-pyrazole-isothiazole-tert-butyl carboxylate-1,1-dioxide forms continuous, homogenous, and bead-free fibers (droplet-like beads are a common problem in electrospinning). We analyzed the compounds and the fibers with various spectroscopic techniques (MS, IR and Raman). Electrospinning does not change chemical composition and configuration, suggesting the monomeric form of the compounds even in the fibers. Interestingly, we found that the stereochemistry of the scaffold can affect the ability of the peptide to be electrospun.

A novel catalytic system based on covalently modified DNA is described.

For tobacco mosaic virus (TMV) as a model virus, this article shows typical issues of scanning soft biological matter by atomic force microscopy (AFM). TMV adsorbed on chemically different flat surfaces, gold, mica, and APDMES-functionalized silicon, is studied in air and aqueous environment. In air, the TMV particles arrangement shows some variety, depending on the substrate. The height of TMV is reduced to 13.7, 15.8, and 15.6 nm, for gold, APDMES, and mica, respectively while the width is about similar to 30 nm due to the influence of the tip radius. In aqueous solution, the surface charges of the virus and the solid support play an important role in the virus adsorption process. While deposition on negatively charged mica is favored only at low pH values, it is shown that positively charged APDMES functionalized silicon can be a suitable substrate to work with at neutral pHs. The effects of cantilever oscillation's free amplitude (A(0)) and the amplitude set-point (A) are also assessed here. While high A(0) prompt reversible deformation of TMV in measurements performed in air, irreversible damage of the virus in liquid conditions (water) is observed using stiff cantilevers (0.35 N m(-1)) and high A(0) (81 nm), leading to a 6 nm reduction in the height of TMV after the first scan. Finally, low values of the amplitude set-point (A/A(0)=0.3), which means applying higher forces to the sample, also brings the damage of TMV virus assemblies, reducing its monolayer roughness to 0.3 nm.

High-resolution microscopy techniques have been extensively used to investigate the structure of soft, biological matter at the nanoscale, from very thin membranes to small objects, like viruses. Electron microscopy techniques allow for obtaining extraordinary resolution by averaging signals from multiple identical structures. In contrast, atomic force microscopy (AFM) collects data from single entities. Here, it is possible to finely modulate the interaction with the samples, in order to be sensitive to their top surface, avoiding mechanical deformations. However, most biological surfaces are highly curved, such as fibers or tubes, and ultimate details of their surface are in the vicinity of steep height variations. This limits lateral resolution, even when sharp probes are used. We overcome this problem by using multifrequency force microscopy on a textbook example, the Tobacco Mosaic Virus (TMV). We achieved unprecedented resolution in local maps of amplitude and phase shift of the second excited mode, recorded together with sample topography. Our data, which combine multifrequency imaging and Fourier analysis, confirm the structure deduced from averaging techniques (XRD, cryoEM) for surface features of single virus particles, down to the helical pitch of the coat protein subunits, 2.3 nm. Remarkably, multifrequency AFM images do not require any image postprocessing.

The adsorption of proteins on surfaces often results in a change of their structural behavior and consequently, a loss of bioactivity. One experimental method to study interactions on a molecular level is single molecular force spectroscopy that permits to measure forces down to the pico-newton range. In this work, the binding force between human serum albumin (HSA), covalently immobilized on glutaraldehyde modified gold substrates, and ibuprofen sodium salt was studied by means of single molecular force spectroscopy. First of all, a protocol was established to functionalize atomic force microscopy (AFM) tips with ibuprofen. The immobilization protocol was additionally tested by quartz crystal microbalance with dissipation (QCM-D) and contact angle measurements. AFM was used to characterize the adsorption of HSA on gold substrates, which lead to a packed monolayer of thickness slightly lower than the reported value in solution. Finally, single molecule spectroscopy results were used to characterize the binding force between albumin and ibuprofen and calculate the distance of the transition state (0.6 nm) and the dissociation rate constant (0.055 s(-1)). The results might indicate that part of the adsorbed protein still preserves its functionality upon adsorption.

Fabrication of novel bio-supramolecular structures was achieved by recrystallizing the bacterial surface protein SbpA on amorphous and semicrystalline polylactide derivatives. Differential scanning calorimetry showed that the glass transition temperature (T(g)) for (poly-L-lactide)-PLLA, poly(L,D-lactide)-PDLLA, poly(lactide-co-glycolide)-PLGA and poly(lactide-co-caprolactone)-PLCL was 63 °C, 53 °C, 49 °C and 15 °C, respectively. Tensile stress-strain tests indicated that PLLA, PLGA, and PDLLA had a glassy behaviour when tested below T(g). The obtained Young modulus were 1477 MPa, 1330 MPa, 1306 MPa, and 9.55 MPa for PLLA, PLGA, PDLLA, and PLCL, respectively. Atomic force microscopy results confirmed that SbpA recrystallized on every polymer substrate exhibiting the native S-layer P4 lattice (a = b = 13 nm, γ = 90°). However, the polymer substrate influenced the domain size of the S-protein crystal, with the smallest size for PLLA (0.011 μm(2)), followed by PDLLA (0.034 μm(2)), and PLGA (0.039 μm(2)), and the largest size for PLCL (0.09 μm(2)). quartz crystal microbalance with dissipation monitoring (QCM-D) measurements indicated that the adsorbed protein mass per unit area (~1800 ng cm(-2)) was independent of the mechanical, thermal, and crystalline properties of the polymer support. The slowest protein adsorption rate was observed for amorphous PLCL (the polymer with the weakest mechanical properties and lowest T(g)). QCM-D also monitored protein self-assembly in solution and confirmed that S-layer formation takes place in three main steps: adsorption, self-assembly, and crystal reorganization. Finally, this work shows that biodegradable polylactide derivatives films are a suitable support to form robust biomimetic S-protein layers.

Biomolecules, cells and the cellular environment have characteristic mechanical properties that determine a range of biological responses. The affected responses include the differentiation and phenotypic expression of cells, an area that has gained prominence due to the current interest in the control of stem cell development. Recent research on biomaterials includes many measurements that have been made on a micro or nano-scale and which are not well described by continuum models. The focus of this review is on the integration and comparison of information obtained from different experimental techniques: mechanical properties are discussed in terms of the wide range of molecular motions and relaxation times that are characteristic of biological materials. Starting at the smaller end of the scale, one component which will be almost universally present in biomolecular samples is water; although bulk water has a relaxation time that would make it fluid in typical experiments, interfacial water and water in confined films will exhibit much slower motions and may therefore show an elastic response, depending on the experimental technique used for the measurements. Water at the surface of hydrophilic solids may thus appear elastic when characterised using high-frequency acoustic devices such as the quartz crystal microbalance (QCM), although the layer will still be fluid in AFM measurements at typical load rates. Likewise, lipid bilayers are viscous at low shear but would be elastic at a sufficiently high frequency. Supported lipid bilayers (SLB) are effectively elastic in acoustic experiments; this could be due to the relaxation time with respect to shear displacements in the bilayer. At the larger end of the size scale, whole cells can also show a frequency-dependent transition to elastic behaviour, at frequencies as low as 0.1 Hz. Other examples mentioned here include proteins and the protein networks of cells.

Bacterial crystalline surface layers (S-layers) are the outermost envelope of prokaryotic organisms representing the simplest biological membranes developed during evolution. In this context, the bacterial protein SbpA has already shown its intrinsic ability to reassemble on different substrates forming protein crystals of square lattice symmetry. In this work, we present the interaction between the bacterial protein SbpA and five self-assembled monolayers carrying methyl (CH3), hydroxyl (OH), carboxylic acid (COOH) and mannose (C6H12O6) as functional groups. Protein adsorption and S-layer formation have been characterized by atomic force microscopy (AFM) while protein adsorption kinetics, mass uptake and the protein layer viscoelastic properties were investigated with quartz crystal microbalance with dissipation monitoring (QCM-D). The results indicate that the protein adsorption rate and crystalline domain area depend on surface chemistry and protein concentration. Furthermore, electrostatic interactions tune different protein rate adsorption and S-layer recrystallization pathways. Electrostatic interactions induce faster adsorption rate than hydrophobic or hydrophilic interactions. Finally, the shear modulus and the viscosity of the recrystallized S-layer on CH3C6S, CH3C11S and COOHC11S substrates were calculated from QCM-D measurements. Protein-protein interactions seem to play a main role in the mechanical stability of the formed protein (crystal) bilayer.

The self-assembly kinetics and nanocrystal formation of the bacterial surface-layer-protein SbpA are studied with a combination of quartz crystal microbalance with dissipation monitoring (QCM-D) and atomic force microscopy (AFM). Silane coupling agents, aminopropyltriethoxysilane (APTS) and octadecyltrichlorosilane (OTS), are used to vary the protein-surface interaction in order to induce new recrystallization pathways. The results show that the final S-layer crystal lattice parameters (a = b = 14 nm, gamma = 90 degrees ), the layer thickness (15 nm), and the adsorbed mass density (1700 ng cm(-2)) are independent of the surface chemistry. Nevertheless, the adsorption rate is five times faster on APTS and OTS than on SiO(2,) strongly affecting protein nucleation and growth. As a consequence, protein crystalline domains of 0.02 microm(2) for APTS and 0.05 microm(2) for OTS are formed, while for silicon dioxide the protein domains have a typical size of about 32 microm(2). In addition, more-rigid crystalline protein layers are formed on hydrophobic substrates. In situ AFM experiments reveal three different kinetic steps: adsorption, self-assembly, and crystalline-domain reorganization. These steps are corroborated by frequency-dissipation curves. Finally, it is shown that protein adsorption is a diffusion-driven process. Experiments at different protein concentrations demonstrate that protein adsorption saturates at 0.05 mg mL(-1) on silane-coated substrates and at 0.07 mg mL(-1) on hydrophilic silicon dioxide.

SmallVolume 6, Issue 3 Frontispiece Nanocrystal formation: Small 3/2010 Aitziber Eleta Lopez, Aitziber Eleta Lopez Biosurfaces Unit, CIC biomaGUNE Paseo Miramón 182, 20009 San Sebastián (Spain)Search for more papers by this authorSusana Moreno-Flores, Susana Moreno-Flores Biosurfaces Unit, CIC biomaGUNE Paseo Miramón 182, 20009 San Sebastián (Spain)Search for more papers by this authorDietmar Pum, Dietmar Pum Department for Nanobiotechnology University of Natural Resources and Applied Life Sciences Gregor-Mendel Strasse 33, Vienna A-1180 (Austria)Search for more papers by this authorUwe B. Sleytr, Uwe B. Sleytr Department for Nanobiotechnology University of Natural Resources and Applied Life Sciences Gregor-Mendel Strasse 33, Vienna A-1180 (Austria)Search for more papers by this authorJosé L. Toca-Herrera, Corresponding Author José L. Toca-Herrera [email protected] Biosurfaces Unit, CIC biomaGUNE Paseo Miramón 182, 20009 San Sebastián (Spain)Biosurfaces Unit, CIC biomaGUNE Paseo Miramón 182, 20009 San Sebastián (Spain)Search for more papers by this author Aitziber Eleta Lopez, Aitziber Eleta Lopez Biosurfaces Unit, CIC biomaGUNE Paseo Miramón 182, 20009 San Sebastián (Spain)Search for more papers by this authorSusana Moreno-Flores, Susana Moreno-Flores Biosurfaces Unit, CIC biomaGUNE Paseo Miramón 182, 20009 San Sebastián (Spain)Search for more papers by this authorDietmar Pum, Dietmar Pum Department for Nanobiotechnology University of Natural Resources and Applied Life Sciences Gregor-Mendel Strasse 33, Vienna A-1180 (Austria)Search for more papers by this authorUwe B. Sleytr, Uwe B. Sleytr Department for Nanobiotechnology University of Natural Resources and Applied Life Sciences Gregor-Mendel Strasse 33, Vienna A-1180 (Austria)Search for more papers by this authorJosé L. Toca-Herrera, Corresponding Author José L. Toca-Herrera [email protected] Biosurfaces Unit, CIC biomaGUNE Paseo Miramón 182, 20009 San Sebastián (Spain)Biosurfaces Unit, CIC biomaGUNE Paseo Miramón 182, 20009 San Sebastián (Spain)Search for more papers by this author First published: 27 January 2010 https://doi.org/10.1002/smll.201090007AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat Graphical Abstract The frontispiece shows four different stages of bacterial nanocrystal formation on 3-aminopropyltriethoxysilane, measured in situ with an atomic force microscope. The growth of protein patches due to individual protein self-assembly can be seen at the border of the crystal patch. Bacterial S-layer proteins are able to self-assemble on many different surfaces to form nanostructured biomimetic crystals. Substrate hydrophobicity affects protein adsorption rate and crystal domain size but has no influence on the protein layer thickness, the crystal-lattice parameters, or the final adsorbed mass density. The S-protein crystal formation occurs in three steps: nucleation, growth (self-assembly), and domain reorganization. For more information, please read the Full Paper 'Surface Dependence of Protein Nanocrystal Formation" by J. L. Toca-Herrera et al., beginning on page 396. Volume6, Issue3February 5, 2010 RelatedInformation