A novel medical device coating prevents Staphylococcus aureus biofilm formation on medical device surfaces.

Prevention of device related infections due to Staphylococcus aureus biofilms on devices represents a significant challenge. Such infections have recently been shown to be dependent on the coagulation pathway via activation of pro-thrombin and fibrin production. Three direct-thrombin inhibitors, argatroban, hirudin and dabigatran, were examined to determine their effect on preventing S. aureus biofilm on plastic biochip surfaces under shear stress using an in vivo relevant model of infection. Surface functionalization of polyurethane discs via dityrosine covalent crosslinking with hirudin was performed and changes in bacterial density and microscopic appearances determined. The three direct-thrombin inhibitors prevented S. aureus biofilm formation on plasma-coated surfaces treated with these agents. Coating of polyurethane with one of these agents, hirudin, significantly inhibited biofilm formation on the modified surface. These findings reveal the exciting potential for coating biomaterial surfaces with direct thrombin inhibitors to prevent staphylococcal binding and subsequent device-related infections.

Atomic view of the histidine environment stabilizing higher-pH conformations of pH-dependent proteins.

External stimuli are powerful tools that naturally control protein assemblies and functions. For example, during viral entry and exit changes in pH are known to trigger large protein conformational changes. However, the molecular features stabilizing the higher pH structures remain unclear. Here we elucidate the conformational change of a self-assembling peptide that forms either small or large nanotubes dependent on the pH. The sub-angstrom high-pH peptide structure reveals a globular conformation stabilized through a strong histidine-serine H-bond and a tight histidine-aromatic packing. Lowering the pH induces histidine protonation, disrupts these interactions and triggers a large change to an extended ß-sheet-based conformation. Re-visiting available structures of proteins with pH-dependent conformations reveals both histidine-containing aromatic pockets and histidine-serine proximity as key motifs in higher pH structures. The mechanism discovered in this study may thus be generally used by pH-dependent proteins and opens new prospects in the field of nanomaterials.

Bacteriophage SPP1 tail tube protein self-assembles into ß-structure-rich tubes.

The majority of known bacteriophages have long tails that serve for bacterial target recognition and viral DNA delivery into the host. These structures form a tube from the viral capsid to the bacterial cell. The tube is formed primarily by a helical array of tail tube protein (TTP) subunits. In phages with a contractile tail, the TTP tube is surrounded by a sheath structure. Here, we report the first evidence that a phage TTP, gp17.1 of siphophage SPP1, self-assembles into long tubes in the absence of other viral proteins. gp17.1 does not exhibit a stable globular structure when monomeric in solution, even if it was confidently predicted to adopt the ß-sandwich fold of phage λ TTP. However, Fourier transform infrared and nuclear magnetic resonance spectroscopy analyses showed that its ß-sheet content increases significantly during tube assembly, suggesting that gp17.1 acquires a stable ß-sandwich fold only after self-assembly. EM analyses revealed that the tube is formed by hexameric rings stacked helicoidally with the same organization and helical parameters found for the tail of SPP1 virions. These parameters were used to build a pseudo-atomic model of the TTP tube. The large loop spanning residues 40-56 is located on the inner surface of the tube, at the interface between adjacent monomers and hexamers. In line with our structural predictions, deletion of this loop hinders gp17.1 tube assembly in vitro and interferes with SPP1 tail assembly during phage particle morphogenesis in bacteria.

Combination of theoretical and experimental approaches for the design and study of fibril-forming peptides.

Self-assembling peptides that can form supramolecular structures such as fibrils, ribbons, and nanotubes are of particular interest to modern bionanotechnology and materials science. Their ability to form biocompatible nanostructures under mild conditions through non-covalent interactions offers a big biofabrication advantage. Structural motifs extracted from natural proteins are an important source of inspiration for the rational design of such peptides. Examples include designer self-assembling peptides that correspond to natural coiled-coil motifs, amyloid-forming proteins, and natural fibrous proteins. In this chapter, we focus on the exploitation of structural information from beta-structured natural fibers. We review a case study of short peptides that correspond to sequences from the adenovirus fiber shaft. We describe both theoretical methods for the study of their self-assembly potential and basic experimental protocols for the assessment of fibril-forming assembly.

Designed self-assembling peptides as templates for the synthesis of metal nanoparticles.

Self-assembling peptides are water soluble and form biocompatible nanostructures under mild conditions through non-covalent interactions. They form supramolecular structures such as ribbons, nanotubes, and fibrils. Of particular interest is the possibility of using these peptide fibrils as templates for the growth of inorganic materials, such as metallic nanoparticles. The ability to reliably produce metal-coated fibrils with robust binding of metal nanoparticles is a vital first step towards the exploitation of these fibrils as conducting nanowires with applications in nano-circuitry. One promising strategy consists of the rational introduction of metal-binding amino acids (such as cysteine) at the level of the peptide building block. Upon assembly of the building blocks into fibrils, cysteine residues that remain accessible at the outside of the fibril core could serve as nucleation sites for metals. We will review in this chapter a case study of rationally designed cysteine-containing peptides and basic protocols for their metallization with silver, gold, and platinum nanoparticles.

Effect of solvent on the self-assembly of dialanine and diphenylalanine peptides.

Diphenylalanine (FF) is a very common peptide with many potential applications, both biological and technological, due to a large number of different nanostructures which it attains. The current work concerns a detailed study of the self-assembled structures of FF in two different solvents, an aqueous (H2O) and an organic (CH3OH) through simulations and experiments. Detailed atomistic molecular dynamics (MD) simulations of FF in both solvents have been performed, using an explicit solvent model. The self-assembling propensity of FF in water is obvious while in methanol a very weak self-assembling propensity is observed. We studied and compared structural properties of FF in the two different solvents and a comparison with a system of dialanine (AA) in the corresponding solvents was also performed. In addition, temperature-dependence studies were carried out. Finally, the simulation predictions were compared to new experimental data, which were produced in the framework of the present work. A very good qualitative agreement between simulation and experimental observations was found.

Silica biotemplating by self-assembling peptides via serine residues activated by the peptide amino terminal group.

Self-assembling biological materials increasingly serve as templates for the binding of inorganic materials and fabrication of composite nanowires, tubes, etc. with important applications in nanobiotechnology. We have previously reported the use of a self-assembling octapeptide building block as scaffold for the systematic introduction of metal-binding residues, namely cysteines, at the first two amino acids within the sequence (Kasotakis et al., Biopolymers 2009, 92, 164-172). We have also reported unexpected behavior of serine within the octapeptide NH2 − NSGAITIG − CONH2( (Asparagine-Serine-Glycine-Alanine-Isoleucine-Threonine-Isoleucine-Glycine) in nucleating gold and platinum nanoparticles. Herein, we report that this serine residue is instrumental in nucleating silica nanoparticles on the surface of the self-assembled fibrils from TEOS (tetraethyl orthosilicate) precursors. We carried out a systematic investigation of the adjacent functionalities and we propose that this serine residue is rendered abnormally nucleophilic through proton abstraction by the N-terminal amino group of the peptide. Peptides with a threonine or a cysteine residue at position 2 are also able to nucleate silica nanoparticles. We propose that rationally designed self-assembling peptides bearing hydroxyl groups adjacent to free amine functionalities could be used for targeted templating of biogenic and even nonbiogenic oxides.

Self-assembly into spheres of a hybrid diphenylalanine-porphyrin: increased fluorescence lifetime and conserved electronic properties.

A series of protected phenylalanine and diphenylalanine derivatives have been coupled through a peptide bond to a monoaminoporphyrin to form new materials. A comparative study in solution and in the solid state has been performed and confirmed new and interesting properties for the self-assembled hybrid materials while conserving the electronic properties of the chromophore. Thus, they are powerful candidates for use in dye-sensitized solar cells.

Electrostatic force microscopy of self-assembled peptide structures.

In this report electrostatic force microscopy (EFM) is used to study different peptide self-assembled structures such as tubes and particles. It is shown that not only geometrical information can be obtained using EFM, but also information about the composition of different structures. In particular we use EFM to investigate the structures of diphenylalanine peptide tubes, particles, and CSGAITIG peptide particles placed on pre-fabricated SiO(2) surfaces with a backgate. We show that the cavity in the peptide tubes could be due to the presence of water residues. Additionally we show that self-assembled amyloid peptides form spherical solid structures containing the same self-assembled peptide in its interior. In both cases transmission electron microscopy is used to verify these structures. Further, the limitations of the EFM technique are discussed, especially when the observed structures become small compared with the radius of the AFM tip used. Finally, an agreement between the detected signal and the structure of the hollow peptide tubes is demonstrated.

Development of an electrochemical metal-ion biosensor using self-assembled peptide nanofibrils.

This article describes the combination of self-assembled peptide nanofibrils with metal electrodes for the development of an electrochemical metal-ion biosensor. The biological nanofibrils were immobilized on gold electrodes and used as biorecognition elements for the complexation with copper ions. These nanofibrils were obtained under aqueous conditions, at room temperature and outside the clean room. The functionalized gold electrode was evaluated by cyclic voltammetry, impedance spectroscopy, energy dispersive X-ray and atomic force microscopy. The obtained results displayed a layer of nanofibrils able to complex with copper ions in solution. The response of the obtained biosensor was linear up to 50 µM copper and presented a sensitivity of 0.68 µA cm⁻² µM⁻¹. Moreover, the fabricated sensor could be regenerated to a copper-free state allowing its reutilization.

Surface-templated fibril growth of peptide fragments from the shaft domain of the adenovirus fibre protein.

Here we present a study of five analogues of a fragment from the shaft domain of the adenovirus fibre protein that readily form fibrils under a range of conditions. Using atomic force microscopy the fibrillisation of these peptides at the liquid/solid interface utilizing ordered crystalline substrates has been investigated. Our results demonstrate that the assembly pathway at the liquid/solid interface enables only the formation of truncated fibrillar structures, which align along the substrate's underlying atomic lattice during growth. Furthermore, that the concentration and volume of solution applied can be used to directly control the density of fibrillar coverage at the surface.

Amyloid-like self-assembly of peptide sequences from the adenovirus fiber shaft: insights from molecular dynamics simulations.

The self-assembly of peptides and proteins into nanostructures is related to the fundamental problems of protein folding and misfolding and has potential applications in medicine, materials science and nanotechnology. Natural peptides, corresponding to sequence repeats from self-assembling proteins, may constitute elementary building blocks of such nanostructures. In this work, we study by implicit-solvent replica-exchange simulations the self-assembly of two amyloidogenic sequences derived from the naturally occurring fiber shaft of the adenovirus, the octapeptide NSGAITIG (asparagine-serine-glycine-alanine-isoleucine-threonine-isoleucine-glycine) and its hexapeptide counterpart, GAITIG. In accordance with their amyloidogenic capacity, both peptides form readily intermolecular beta-sheets, stabilized by extensive main- and side-chain contacts involving the C-terminal moieties (segments 3-8 and 2-6, respectively). The structural and energetic properties of these sheets are analyzed extensively. The N-terminal residues Asn1 and Ser2 of the octapeptide remain disordered in the sheets, suggesting that these residues are exposed at the exterior of the fibrils and accessible. On the basis of insight provided by the simulations, cysteine residues were recently substituted at positions 1 and 2 of NSGAITIG; the newly designed peptides maintain their amyloidogenic properties and can bind to silver, gold and platinum nanoparticles [Kasotakis et al. Biopolymers 2009, 92, 164-172]. Computational investigation can identify suitable positions for rational modification of peptide building blocks, aiming at the fabrication of novel biomaterials.

Design of metal-binding sites onto self-assembled peptide fibrils.

The ability to develop a rational basis for the binding of inorganic materials to specific binding sites within self-assembling biological scaffolds has important applications in nanobiotechnology. Amyloid-forming peptides are a class of such scaffolds and show enormous potential as templates for the fabrication of low resistance, conducting nanowires. Here we report the use of a self-assembling peptide building block as scaffold for the systematic introduction of metal-binding residues at specific locations within the structure. The octapeptide NSGAITIG (Asparagine-Serine-Glycine-Alanine-Isoleucine-Threonine-Isoleucine-Glycine) from the fiber protein of adenovirus has been identified in previous structural studies as an elementary fibril-forming building block. Using this building block as a scaffold, we have designed three new cysteine-containing octa-peptides to study their eventual fibril-forming ability and potential templating of metal nanoparticles. We find that the cysteine substitutions do not alter the fibril-forming potential of the peptides, and that the fibrils formed bind efficiently to silver, gold, and platinum nanoparticles; furthermore, we report unexpected behavior of serine in nucleating gold and platinum nanoparticles. We find that combination of cysteine and serine residues projecting from adjacent sites on a peptide scaffold represents a potentially useful strategy in nucleating inorganic materials. The ability to reliably produce metal-coated fibrils is a vital first step towards the exploitation of these fibrils as conducting nanowires with applications in nano-circuitry. Short, biologically inspired self-assembling peptide scaffolds derived from natural fibrous proteins with known three-dimensional structure may provide a viable approach towards the rational design of inorganic nanowires.

Directed three-dimensional patterning of self-assembled peptide fibrils.

Molecular self-assembly is emerging as a viable "bottom-up" approach for fabricating nanostructures. Self-assembled biomolecular structures are particularly attractive, due to their versatile chemistry, molecular recognition properties, and biocompatibility. Among them, amyloid protein and peptide fibrils are self-assembled nanostructures with unique physical and chemical stability, formed from quite simple building blocks; their ability to work as a template for the fabrication of low resistance, conducting nanowires has already been demonstrated. The precise positioning of peptide-based nanostructures is an essential part of their use in technological applications, and their controlled assembly, positioning, and integration into microsystems is a problem of considerable current interest. To date, their positioning has been limited to their placement on flat surfaces or to the fabrication of peptide arrays. Here, we propose a new method for the precise, three-dimensional patterning of amyloid fibrils. The technique, which combines femtosecond laser technology and biotin-avidin mediated assembly on a polymeric matrix, can be applied in a wide variety of fields, from molecular electronics to tissue engineering.