Automated detection of protein unfolding events in atomic force microscopy force curves.

Atomic force microscopy is not only a high-resolution imaging device but also a mechanical machine, which can be used either to indent or stretch (soft) biomaterials. Due to the statistical nature of such materials (i.e., hydrogels or polymers) hundreds of force-distance curves are required to describe their mechanical properties. In this manuscript, we present an automated system for polymer unfolding detection based on continuous wavelet analysis. We have tested the automated program on elastin, which is an important protein that provides elasticity to tissues and organs. Our results show that elastin changes its mechanical behavior in the presence of electrolytes. In particular, we show that NaCl has a different effect on the contour length than CaCl2 for similar unfolding forces. In addition, we provide the program in the supporting information for the researches facing such kind of problem.

A stereochemical switch in the aDrs model system, a candidate for a functional amyloid.

Amyloid fibrils are commonly observed to adopt multiple distinct morphologies, which eventually can have significantly different neurotoxicities, as e.g. demonstrated in case of the Alzheimer peptide. The architecture of amyloid deposits is apparently also determined by the stereochemistry of amino acids. Post-translational changes of the chirality of certain residues may thus be a factor in controlling the formation of functional or disease-related amyloids. Anionic dermaseptin (aDrs), an unusual peptide from the skin secretions of the frog Pachymedusa dacnicolor, assembles to amyloid-like fibrils in a pH-dependent manner, which could play a functional role in defense. aDrs can be enzymatically converted into the diastereomer [d-Leu2]-aDrs by an l/d-isomerase. EM and AFM on fibrils formed by these isomers have shown that their predominant morphology is controlled by the stereochemistry of residue 2, whereas kinetic and thermodynamic parameters of aggregation are barely affected. When fibrils were grown from preformed seeds, backbone stereochemistry rather than templating-effects apparently dominated the superstructural organization of the isomers. Interestingly, MD indicated small differences in the conformational propensities between the isomers. Our results demonstrate how d-amino acid substitutions could take active part in the formation of functional or disease-related amyloid. Moreover, these findings contribute to the development of amyloid-based nanomaterials.

Self-assembly of S-layer-enveloped cytochrome c polyelectrolyte multilayers.

We report a study of the electrostatic layer-by-layer self-assembly of electroactive polyelectrolyte multilayers incorporating the redox protein cytochrome c (cyt c) combined with recrystallization of the bacterial cell wall surface layer from Bacillus sphaericus CCM 2177 SbpA (S-layer). The polyelectrolyte multilayer assembly was prepared on flat gold electrodes with a nanometer-scale roughness that allowed monitoring of the film formation throughout all the assembly stages by atomic force microscopy measurements in liquid with respect to topography and forces. The deposition of alternating layers of sulfonated polyaniline and cyt c was carried out by adsorption from the corresponding solutions on a cyt c monolayer electrode. The electroactivity of cyt c within the assembly was confirmed by cyclic voltammetry. We showed that the surface properties of the electrode terminating layer change after each adsorption step accordingly. We also found that S-layer recrystallization on the top of the multilayer film was feasible while electroactivity of cyt c within a polyelectrolyte matrix was partially maintained. This approach offers a new strategy to design a biocompatible and permselective outer envelope of a polyelectrolyte multilayer, promising sensor applications.

Novel biocatalysts based on S-layer self-assembly of Geobacillus stearothermophilus NRS 2004/3a: a nanobiotechnological approach.

The crystalline cell-surface (S) layer sgsE of Geobacillus stearothermophilus NRS 2004/3a represents a natural protein self-assembly system with nanometer-scale periodicity that is evaluated as a combined carrier/patterning element for the conception of novel types of biocatalyst aiming at the controllable display of biocatalytic epitopes, storage stability, and reuse. The glucose-1-phosphate thymidylyltransferase RmlA is used as a model enzyme and chimeric proteins are constructed by translational fusion of rmlA to the C-terminus of truncated forms of sgsE (rSgsE (131-903), rSgsE(331-903)) and used for the construction of three principal types of biocatalysts: soluble (monomeric), self-assembled in aqueous solution, and recrystallized on negatively charged liposomes. Enzyme activity of the biocatalysts reaches up to 100 % compared to sole RmlA cloned from the same bacterium. The S-layer portion of the biocatalysts confers significantly improved shelf life to the fused enzyme without loss of activity over more than three months, and also enables biocatalyst recycling. These nanopatterned composites may open up new functional concepts for biocatalytic applications in nanobiotechnology.

Chemical and thermal denaturation of crystalline bacterial S-layer proteins: an atomic force microscopy study.

Crystalline monomolecular cell surface layers, S-layers, are one of the most common outermost cell envelope components of the prokaryotic organisms (bacteria and archaeda) that protects them from competitive habitats. Since isolated S-protein subunits are able to re-assemble into crystalline arrays on lipid films and solid supports making biomimetic surfaces, S-layer technology is currently used in nanobiotechnology. An important aspect of the biomimetic surfaces built with S-layers is their stability under extreme solvent conditions or temperature. Chemical (pH, alcohol) and physical (thermal) denaturant conditions were employed to test the stability of S-layers. Recrystallized bacterial surface layers from Bacillus sphaericus (SbpA) on hydrophilic silicon wafers loses the crystalline structure at 80% ethanol/water mixtures, the change in structure being reversible after treating the surface with buffer solution. SbpA on silicon supports denatures at pH 3 and at 70 degrees C, and the process is irreversible. Cross-linking of SbpA enhances the stability for high ethanol and acidic conditions, but it does not improve thermal stability. Recrystallized SbpA on secondary cell wall polymer (SCWP), a natural environment for the protein layer, is more resistant to ethanol and pH exposure than recrystallized SbpA on hydrophilic silicon supports. Atomic force microscopy (AFM) was used to monitor the loss of stability and the changes in protein layer conformation.