Protein fibrillogenesis model tracked by its intrinsic time-resolved emission spectra.

The excited-state kinetics of the fluorescence of tyrosine in a de novo protein fibrillogenesis model was investigated as a potential tool for monitoring protein fibre formation and complexation with glucose (glycation). In stark contrast to insulin the time-resolved emission spectra (TRES) recorded over the period of 700 hours in buffered solutions of the model with and without glucose revealed no apparent changes in Tyr fluorescence responses. This indicates the stability of the model and provides a measurement-supported basis for its use as a reference material in fluorescence studies of protein aggregation.

Imaging Protein Fibers at the Nanoscale and In Situ.

Protein self-assembly offers a rich repertoire of tools and technologies. However, despite significant progress in this area, a deterministic measure of the phenomenon, which might lead to predictable relationships between protein components, assembly mechanisms, and ultimately function, is lacking. Often the challenge relates to the choice of the most informative and precise measurements that can link the chemistry of the building blocks with the resulting assembly, ideally in situ and in real time. Using the example of protein fibrillogenesis-a self-assembly process fundamental to nearly every aspect of biological organization, from viral assembly to tissue restoration-this chapter demonstrates how protein self-assembly can be visually and precisely measured while providing measurement protocols applicable to other self-assembly systems.

Antimicrobial peptide capsids of de novo design.

The spread of bacterial resistance to antibiotics poses the need for antimicrobial discovery. With traditional search paradigms being exhausted, approaches that are altogether different from antibiotics may offer promising and creative solutions. Here, we introduce a de novo peptide topology that-by emulating the virus architecture-assembles into discrete antimicrobial capsids. Using the combination of high-resolution and real-time imaging, we demonstrate that these artificial capsids assemble as 20-nm hollow shells that attack bacterial membranes and upon landing on phospholipid bilayers instantaneously (seconds) convert into rapidly expanding pores causing membrane lysis (minutes). The designed capsids show broad antimicrobial activities, thus executing one primary function-they destroy bacteria on contact.

CREIM: Coffee Ring Effect Imaging Model for Monitoring Protein Self-Assembly in Situ.

Protein self-assembly is fundamental to nanotechnology. Self-assembling structures are produced under static in vitro conditions typically forming over hours. In contrast, hydrodynamic intracellular environments employ far shorter time scales to compartmentalize highly concentrated protein solutions. Herein, we exploit the radial capillary flow within a drying sessile droplet (the coffee ring effect) to emulate dynamic native environments and monitor an archetypal protein assembly in situ using high-speed super-resolution imaging. We demonstrate that the assembly can be empirically driven to completion within minutes to seconds without apparent changes in supramolecular morphology. The model offers a reliable tool for the diagnosis and engineering of self-assembling systems under nonequilibrium conditions.

Linear and orthogonal peptide templating of silicified protein fibres.

Biomineralisation is essential for biology. Specialist proteins use peptide motifs that catalyse mineral deposition into nano-to-microscale inorganic materials. Unlike in native proteins, the motifs incorporated into self-assembled fibres can persistently propagate on the microscopic scale enabling empirically defined silica nanostructures. Herein we show that the two main modes of motif templating - linear and orthogonal - in self-assembling, fibre-forming peptide sequences effectively silicify protein fibres. We show that the mere charge and morphology of protein fibres are not sufficient for silica deposition, but it is the synergy between fibrillogenesis and silica-specific motifs regularly spaced in fibres that ensures silica templating, regardless of the relative orientation of the motifs.

Binary Encoding of Random Peptide Sequences for Selective and Differential Antimicrobial Mechanisms.

Binary encoding of peptide sequences into differential antimicrobial mechanisms is reported. Such sequences are random in composition, but controllable in chain length, are assembled from the same two amino acids, but differ in the stereochemistry of one. Regardless of chirality, the sequences lyse bacteria including the "superbugs" methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE). Sequences with the same chirality, so-called homochiral sequences, assemble into antimicrobial pores and form contiguous helices that are biologically promiscuous and hemolytic. By contrast, heterochiral sequences that lack such persistence selectively attack bacterial membranes without oligomerizing into visible pores. These results offer a mechanistic rationale for designing membrane-selective and sequence-independent antimicrobials.

Engineering monolayer poration for rapid exfoliation of microbial membranes.

The spread of bacterial resistance to traditional antibiotics continues to stimulate the search for alternative antimicrobial strategies. All forms of life, from bacteria to humans, are postulated to rely on a fundamental host defense mechanism, which exploits the formation of open pores in microbial phospholipid bilayers. Here we predict that transmembrane poration is not necessary for antimicrobial activity and reveal a distinct poration mechanism that targets the outer leaflet of phospholipid bilayers. Using a combination of molecular-scale and real-time imaging, spectroscopy and spectrometry approaches, we introduce a structural motif with a universal insertion mode in reconstituted membranes and live bacteria. We demonstrate that this motif rapidly assembles into monolayer pits that coalesce during progressive membrane exfoliation, leading to bacterial cell death within minutes. The findings offer a new physical basis for designing effective antibiotics.

Modulating charge-dependent and folding-mediated antimicrobial interactions at peptide-lipid interfaces.

Peptide-lipid interactions support a variety of biological functions. Of particular interest are those that underpin fundamental mechanisms of innate immunity that are programmed in host defense or antimicrobial peptide sequences found virtually in all multicellular organisms. Here we synthetically modulate antimicrobial peptide-lipid interactions using an archetypal helical antimicrobial peptide and synthetic membranes mimicking bacterial and mammalian membranes in solution. We probe these interactions as a function of membrane-induced folding, membrane stability and peptide-lipid ratios using a correlative approach encompassing light scattering and spectroscopy measurements such as circular dichroism spectroscopy, fluorescence and nuclear magnetic resonance spectroscopy. The peptide behavior is assessed against that of its anionic counterpart having similar propensities for α-helical folding. The results indicate strong correlations between peptide folding and membrane type, supporting folding-responsive binding of antimicrobial peptides to bacterial membranes. The study provides a straightforward approach for modulating structure-activity relationships in the context of membrane-induced antimicrobial action, thus holding promise for the rational design of potent antimicrobial agents.

Peptide engineered microcantilevers for selective chemical force microscopy and monitoring of nanoparticle capture.

Engineered peptides capable of binding to silica have been used to provide contrast in chemical force microscopy and tested for their capacity to selectively capture silica nanoparticles (NPs). Gold coated atomic force microscopy (AFM) microcantilevers with integrated tips and colloidal probes were functionalized with engineered peptides through a thiol group of a terminal cysteine which was linked via a glycine trimer to a 12-mer binding sequence. The functionalized probes demonstrated a significantly increased binding force on silicon oxide areas of a gold-patterned silicon wafer, whereas plain gold probes, and those functionalized with a random permutation of the silica binding peptide motif or an all-histidine sequence displayed similar adhesion forces to gold and silicon oxide. As the functionalized probes also allowed contact mode imaging subsequently to the adhesion mapping, also the associated friction contrast was measured and found to be similar to the adhesion contrast. Furthermore, the adsorption of silica NPs onto planar gold surfaces functionalized in the same manner was observed to be selective. Notably, the surface coverage with silica NPs was found to decrease with increasing pH, implying the importance of electrostatic interactions between the peptide and the NPs. Finally, the adsorption of silica NPs was monitored via the decrease in fundamental resonance frequency of an AFM microcantilever functionalized with silica binding peptides.

Nano-mechanical single-cell sensing of cell-matrix contacts.

Extracellular protein matrices provide a rigidity interface exhibiting nano-mechanical cues that guide cell growth and proliferation. Cells sense such cues using actin-rich filopodia extensions which encourage favourable cell-matrix contacts to recruit more actin-mediated local forces into forming stable focal adhesions. A challenge remains in identifying and measuring these local cellular forces and in establishing empirical relationships between them, cell adhesion and filopodia formation. Here we investigate such relationships using a micromanipulation system designed to operate at the time scale of focal contact dynamics, with the sample frequency of a force probe being 0.1 ms, and to apply and measure forces at nano-to-micro Newton ranges for individual mammalian cells. We explore correlations between cell biomechanics, cell-matrix attachment forces and the spread areas of adhered cells as well as their relative dependence on filopodia formation using synthetic protein matrices with a proven ability to induce enhanced filopodia numbers in adherent cells. This study offers a basis for engineering exploitable cell-matrix contacts in situ at the nanoscale and single-cell levels.

Structurally plastic peptide capsules for synthetic antimicrobial viruses.

A conceptual design for artificial antimicrobial viruses is described. The design emulates viral assembly and function to create self-assembling peptide capsules that promote efficient gene delivery and silencing in mammalian cells. Unlike viruses, however, the capsules are antimicrobial, which allows them to exhibit a dual biological function: gene transport and antimicrobial activity. Unlike other antimicrobials, the capsules act as pre-concentrated antimicrobial agents that elicit rapid and localised membrane-disrupting responses by converting into individual pores at their precise landing positions on membranes. The concept holds promise for engineering virus-like scaffolds with biologically tuneable properties.

Application of Asymmetric Flow Field-Flow Fractionation hyphenations for liposome-antimicrobial peptide interaction.

Asymmetric Flow Field-Flow Fractionation (AF4) combined with multidetector analysis form a promising technique in the field of nanoparticle characterization. This system is able to measure the dimensions and physicochemical properties of nanoparticles with unprecedented accuracy and precision. Here, for the first time, this technique is optimized to characterize the interaction between an archetypal antimicrobial peptide and synthetic membranes. By using charged and neutral liposomes it is possible to mimic some of the charge characteristics of biological membranes. The use of AF4 system allows determining, in a single analysis, information regarding the selectivity of the peptides, the quantity of peptides bound to each liposome, the induced change in the size distribution and morphology of the liposomes. The results obtained provide relevant information for the study of structure-activity relationships in the context of membrane-induced antimicrobial action. This information will contribute to the rational design of potent antimicrobial agents in the future. Moreover, the application of this method to other liposome systems is straightforward and would be extremely useful for a comprehensive characterization with regard to size distribution and protein interaction in the nanomedicine field.

Supramolecular amphipathicity for probing antimicrobial propensity of host defence peptides.

Host defence peptides (HDPs) are effector components of innate immunity that provide defence against pathogens. These are small-to-medium sized proteins which fold into amphipathic conformations toxic to microbial membranes. Here we explore the concept of supramolecular amphipathicity for probing antimicrobial propensity of HDPs using elementary HDP-like amphiphiles. Such amphiphiles are individually inactive, but when ordered into microscopic micellar assemblies, respond to membrane binding according to the orthogonal type of their primary structure. The study demonstrates that inducible supramolecular amphipathicity can discriminate against bacterial growth and colonisation thereby offering a physico-chemical rationale for tuneable targeting of biological membranes.

Filming protein fibrillogenesis in real time.

Protein fibrillogenesis is a universal tool of nano-to-micro scale construction supporting different forms of biological function. Its exploitable potential in nanoscience and technology is substantial, but the direct observation of homogeneous fibre growth able to underpin a kinetic-based rationale for building customized nanostructures in situ is lacking. Here we introduce a kinetic model of de novo protein fibrillogenesis which we imaged at the nanoscale and in real time, filmed. The model helped to reveal that, in contrast to heterogeneous amyloid assemblies, homogeneous protein recruitment is principally characterized by uniform rates of cooperative growth at both ends of growing fibers, bi-directional growth, with lateral growth arrested at a post-seeding stage. The model provides a foundation for in situ engineering of sequence-prescribed fibrous architectures.

Differentially instructive extracellular protein micro-nets.

An ability to construct biological matter from the molecule up holds promise for applications ranging from smart materials to integrated biophysical models for synthetic biology. Biomolecular self-assembly is an efficient strategy for biomaterial construction which can be programmed to support desired function. A challenge remains in replicating the strategy synthetically, that is at will, and differentially, that is for a specific function at a given length scale. Here we introduce a self-assembly topology enabling a net-like architectural mimetic of native extracellular matrices capable of differential responses to cell adhesion--enhanced mammalian cell attachment and proliferation, and enhanced resistance to bacterial colonization--at the native sub-millimeter length scales. The biological performance of such protein micro-nets directly correlates with their morphological and chemical properties, offering thus an application model for differential extracellular matrices.

Arbitrary self-assembly of peptide extracellular microscopic matrices.

Two faces for one matrix: A single bifaceted cyclopeptide block forms highly branched, porous, and intricate fibrillar networks, which span microscopic dimensions and mimic the extracellular matrix to support cell growth and proliferation. The peptide block has two domains connected with triglycine linkers (GGG); the domains consist of positively (blue) and negatively (red) charged heptads that provide interactions between different blocks.

Assessment of structurally diverse philanthotoxin analogues for inhibitory activity on ionotropic glutamate receptor subtypes: discovery of nanomolar, nonselective, and use-dependent antagonists.

An array of analogues of the wasp toxin philanthotoxin-433, in which the asymmetric polyamine moiety was exchanged for spermine and the headgroup replaced with a variety of structurally diverse moieties, was prepared using parallel solid-phase synthesis approaches. In three analogues, the spermine moiety was extended with an amino acid tail, six compounds contained an N-acylated cyclohexylalanine, and four analogues were based on a novel diamino acid design with systematically changed spacer length between N-cyclohexylcarbonyl and N-phenylacetyl substituents. The analogues were studied using two-electrode voltage-clamp electrophysiology employing Xenopus laevis oocytes expressing GluA1(i) AMPA or GluN1/2A NMDA receptors. Several of the analogues showed significantly increased inhibition of the GluN1/2A NMDA receptor. Thus, an analogue containing N-(1-naphtyl)acetyl group showed an IC(50) value of 47 nM. For the diamino acid-based analogues, the optimal spacer length between two N-acyl groups was determined, resulting in an analogue with an IC(50) value of 106 nM.

Conformationally constrained mimetics of laminin peptide YIGSR as precursors for antimetastatic disintegrins.

Conformationally constrained mimetics of the laminin cell-adhesion site, YIGSR, are described. The site is the natural antagonist of the integrin-associated laminin receptor 1 (LAMR1) known to mediate metastatic tumor adhesion. The attachment of selected metastatic cell lines toward the constrained antagonists has been assessed. Observed differential responses prompted by folding preferences of the mimetics revealed stronger attachment activities for turnlike structures. The results permit the conformational design of antimetastatic disintegrins.

Modular design of peptide fibrillar nano- to microstructures.

A general concept for designing self-assembling peptide fibrillar nano- to microstructures is described. The approach relies on the use of straightforward alpha-helical one-heptad (approximately 1 nm) modules without the need for more-specific designs. Various combinations of the modules gave a variety of constructs that were examined by a combination of spectroscopy and microscopy. These show that simple rearrangements and stereochemical conversions of the modules within similar templates lead to different fiber morphologies. The concept opens new strategies for designing fibrous materials with tunable properties at the nanoscale.