Antimicrobial efficacy of cyclic α- and ß-peptides incorporated in polyurethane coatings.

Microbial growth on surfaces poses health concerns and can accelerate the biodegradation of engineered materials and coatings. Cyclic peptides are promising agents to combat biofouling because they are more resistant to enzymatic degradation than their linear counterparts. They can also be designed to interact with extracellular targets and intracellular targets and/or self-assemble into transmembrane pores. Here, we determine the antimicrobial efficacy of two pore-forming cyclic peptides, α-K3W3 and ß-K3W3, against bacterial and fungal liquid cultures and their capacity to inhibit biofilm formation on coated surfaces. These peptides display identical sequences, but the additional methylene group in the peptide backbone of ß-amino acids results in a larger diameter and an enhancement in the dipole moment. In liquid cultures, ß-K3W3 exhibited lower minimum inhibitory concentration values and greater microbicidal power in reducing the number of colony forming units (CFUs) when exposed to a gram-positive bacterium, Staphylococcus aureus, and two fungal strains, Naganishia albida and Papiliotrema laurentii. To evaluate the efficacy against the formation of fungal biofilms on painted surfaces, cyclic peptides were incorporated into polyester-based thermoplastic polyurethane. The formation of N. albida and P. laurentii microcolonies (105 per inoculation) for cells extracted from coatings containing either peptide could not be detected after a 7-day exposure. Moreover, very few CFUs (∼5) formed after 35 days of repeated depositions of freshly cultured P. laurentii every 7 days. In contrast, the number of CFUs for cells extracted from the coating without cyclic peptides was >8 log CFU.

Effect of Water on the Mechanical Properties of Cyclic Peptide Polymers.

Biomaterials are an important source of inspiration for the development of strong and tough materials. Many improved and optimized synthetic materials have been recently developed utilizing this bioinspiration concept. Using side-chain-to-side-chain polymerization of cyclic ß-peptide rings, a novel class of nanomaterials was recently introduced with outstanding mechanical properties such as toughness values greater than natural silks. In this work, molecular dynamics is used to understand the mechanics of side-chain-to-side-chain polymerization of cyclic ß-peptide rings. Unbiased steered molecular dynamics simulations are used to show the difference in the strength of polymerized and unpolymerized processing of similar cyclic rings. The simulations are performed both in aqueous and vacuum environments to capture the role of water on the mechanical properties of the cyclic peptides. Our results show that unpolymerized peptides behave like brittle material, whereas polymerized ones can withstand some stress after initial failure with large values of strain-to-failure. Finally, we have shown that the strength of cyclic peptides in water is higher than in a vacuum.

Pressure cycling technology for challenging proteomic sample processing: application to barnacle adhesive.

Successful proteomic characterization of biological material depends on the development of robust sample processing methods. The acorn barnacle Amphibalanus amphitrite is a biofouling model for adhesive processes, but the identification of causative proteins involved has been hindered by their insoluble nature. Although effective, existing sample processing methods are labor and time intensive, slowing progress in this field. Here, a more efficient sample processing method is described which exploits pressure cycling technology (PCT) in combination with protein solvents. PCT aids in protein extraction and digestion for proteomics analysis. Barnacle adhesive proteins can be extracted and digested in the same tube using PCT, minimizing sample loss, increasing throughput to 16 concurrently processed samples, and decreasing sample processing time to under 8 hours. PCT methods produced similar proteomes in comparison to previous methods. Two solvents which were ineffective at extracting proteins from the adhesive at ambient pressure (urea and methanol) produced more protein identifications under pressure than highly polar hexafluoroisopropanol, leading to the identification and description of >40 novel proteins at the interface. Some of these have homology to proteins with elastomeric properties or domains involved with protein-protein interactions, while many have no sequence similarity to proteins in publicly available databases, highlighting the unique adherent processes evolved by barnacles. The methods described here can not only be used to further characterize barnacle adhesive to combat fouling, but may also be applied to other recalcitrant biological samples, including aggregative or fibrillar protein matrices produced during disease, where a lack of efficient sample processing methods has impeded advancement. Data are available via ProteomeXchange with identifier PXD012730.

Molecular Recognition of Structures Is Key in the Polymerization of Patterned Barnacle Adhesive Sequences.

The permanent adhesive produced by adult barnacles is held together by tightly folded proteins that form amyloid-like materials distinct among marine foulants. In this work, we link stretches of alternating charged and noncharged linear sequences from a family of adhesive proteins to their role in forming fibrillar nanomaterials. Using recombinant proteins and short barnacle cement derived peptides (BCPs), we find a central sequence with charged motifs of the pattern [Gly/Ser/Val/Thr/Ala-X], where X are charged amino acids, to exert specific control over timing, structure, and morphology of fibril formation. While most BCPs remain dormant, the core segment demonstrates rapid polymerization as well as an ability to template other peptides with no propensity for self-assembly. Patterned charge domains assemble dormant peptides through a specific antiparallel ß-sheet structure as measured by FTIR. While charged domains favor an antiparallel structure, BCPs without charged domains switch fibril assembly to favor simpler parallel ß-sheet aggregates. In addition to activation, charged domains direct nanofibers to grow into discrete microns long fibrils similar to the natural adhesive, while segments without such domains only form short branched aggregates. The assembly of adhesive sequences through recognition of structured templates outlines a strategy used by barnacles to control physical mechanisms of underwater adhesive delivery, activation, and curing based on molecular recognition between proteins.

High-performance nanomaterials formed by rigid yet extensible cyclic ß-peptide polymers.

Organisms have evolved biomaterials with an extraordinary convergence of high mechanical strength, toughness, and elasticity. In contrast, synthetic materials excel in stiffness or extensibility, and a combination of the two is necessary to exceed the performance of natural biomaterials. We bridge this materials property gap through the side-chain-to-side-chain polymerization of cyclic ß-peptide rings. Due to their strong dipole moments, the rings self-assemble into rigid nanorods, stabilized by hydrogen bonds. Displayed amines serve as functionalization sites, or, if protonated, force the polymer to adopt an unfolded conformation. This molecular design enhances the processability and extensibility of the biopolymer. Molecular dynamics simulations predict stick-slip deformations dissipate energy at large strains, thereby, yielding toughness values greater than natural silks. Moreover, the synthesis route can be adapted to alter the dimensions and displayed chemistries of nanomaterials with mechanical properties that rival nature.

Oxidase Activity of the Barnacle Adhesive Interface Involves Peroxide-Dependent Catechol Oxidase and Lysyl Oxidase Enzymes.

Oxidases are found to play a growing role in providing functional chemistry to marine adhesives for the permanent attachment of macrofouling organisms. Here, we demonstrate active peroxidase and lysyl oxidase enzymes in the adhesive layer of adult Amphibalanus amphitrite barnacles through live staining, proteomic analysis, and competitive enzyme assays on isolated cement. A novel full-length peroxinectin (AaPxt-1) secreted by barnacles is largely responsible for oxidizing phenolic chemistries; AaPxt-1 is driven by native hydrogen peroxide in the adhesive and oxidizes phenolic substrates typically preferred by phenoloxidases (POX) such as laccase and tyrosinase. A major cement protein component AaCP43 is found to contain ketone/aldehyde modifications via 2,4-dinitrophenylhydrazine (DNPH) derivatization, also called Brady's reagent, of cement proteins and immunoblotting with an anti-DNPH antibody. Our work outlines the landscape of molt-related oxidative pathways exposed to barnacle cement proteins, where ketone- and aldehyde-forming oxidases use peroxide intermediates to modify major cement components such as AaCP43.

Evaluating protocols and analytical methods for peptide adsorption experiments.

This paper evaluates analytical techniques that are relevant for performing reliable quantitative analysis of peptide adsorption on surfaces. Two salient problems are addressed: determining the solution concentrations of model GG-X-GG, X5, and X10 oligopeptides (G = glycine, X = a natural amino acid), and quantitative analysis of these peptides following adsorption on surfaces. To establish a uniform methodology for measuring peptide concentrations in water across the entire GG-X-GG and X n series, three methods were assessed: UV spectroscopy of peptides having a C-terminal tyrosine, the bicinchoninic acid (BCA) protein assay, and amino acid (AA) analysis. Due to shortcomings or caveats associated with each of the different methods, none were effective at measuring concentrations across the entire range of representative model peptides. In general, reliable measurements were within 30% of the nominal concentration based on the weight of as-received lyophilized peptide. In quantitative analysis of model peptides adsorbed on surfaces, X-ray photoelectron spectroscopy (XPS) data for a series of lysine-based peptides (GGKGG, K5, and K10) on Au substrates, and for controls incubated in buffer in the absence of peptides, suggested a significant presence of aliphatic carbon species. Detailed analysis indicated that this carbonaceous contamination adsorbed from the atmosphere after the peptide deposition. The inferred adventitious nature of the observed aliphatic carbon was supported by control experiments in which substrates were sputter-cleaned by Ar(+) ions under ultra-high vacuum (UHV) then re-exposed to ambient air. In contrast to carbon contamination, no adventitious nitrogen species were detected on the controls; therefore, the relative surface densities of irreversibly-adsorbed peptides were calculated by normalizing the N/Au ratios by the average number of nitrogen atoms per residue.