Fabrication of hierarchical hybrid structures using bio-enabled layer-by-layer self-assembly.

Development of versatile and flexible assembly systems for fabrication of functional hybrid nanomaterials with well-defined hierarchical and spatial organization is of a significant importance in practical nanobiotechnology applications. Here we demonstrate a bio-enabled self-assembly technique for fabrication of multi-layered protein and nanometallic assemblies utilizing a modular gold-binding (AuBP1) fusion tag. To accomplish the bottom-up assembly we first genetically fused the AuBP1 peptide sequence to the C'-terminus of maltose-binding protein (MBP) using two different linkers to produce MBP-AuBP1 hetero-functional constructs. Using various spectroscopic techniques, surface plasmon resonance (SPR) and localized surface plasmon resonance (LSPR), we verified the exceptional binding and self-assembly characteristics of AuBP1 peptide. The AuBP1 peptide tag can direct the organization of recombinant MBP protein on various gold surfaces through an efficient control of the organic-inorganic interface at the molecular level. Furthermore using a combination of soft-lithography, self-assembly techniques and advanced AuBP1 peptide tag technology, we produced spatially and hierarchically controlled protein multi-layered assemblies on gold nanoparticle arrays with high molecular packing density and pattering efficiency in simple, reproducible steps. This model system offers layer-by-layer assembly capability based on specific AuBP1 peptide tag and constitutes novel biological routes for biofabrication of various protein arrays, plasmon-active nanometallic assemblies and devices with controlled organization, packing density and architecture.

Engineered Chimeric Peptides as Antimicrobial Surface Coating Agents toward Infection-Free Implants.

Prevention of bacterial colonization and consequent biofilm formation remains a major challenge in implantable medical devices. Implant-associated infections are not only a major cause of implant failures but also their conventional treatment with antibiotics brings further complications due to the escalation in multidrug resistance to a variety of bacterial species. Owing to their unique properties, antimicrobial peptides (AMPs) have gained significant attention as effective agents to combat colonization of microorganisms. These peptides have been shown to exhibit a wide spectrum of activities with specificity to a target cell while having a low tendency for developing bacterial resistance. Engineering biomaterial surfaces that feature AMP properties, therefore, offer a promising approach to prevent implant infections. Here, we engineered a chimeric peptide with bifunctionality that both forms a robust solid-surface coating while presenting antimicrobial property. The individual domains of the chimeric peptides were evaluated for their solid-binding kinetics to titanium substrate as well as for their antimicrobial properties in solution. The antimicrobial efficacy of the chimeric peptide on the implant material was evaluated in vitro against infection by a variety of bacteria, including Streptococcus mutans, Staphylococcus. epidermidis, and Escherichia coli, which are commonly found in oral and orthopedic implant related surgeries. Our results demonstrate significant improvement in reducing bacterial colonization onto titanium surfaces below the detectable limit. Engineered chimeric peptides with freely displayed antimicrobial domains could be a potential solution for developing infection-free surfaces by engineering implant interfaces with highly reduced bacterial colonization property.

Effect of molecular conformations on the adsorption behavior of gold-binding peptides.

Despite extensive recent reports on combinatorially selected inorganic-binding peptides and their bionanotechnological utility as synthesizers and molecular linkers, there is still only limited knowledge about the molecular mechanisms of peptide binding to solid surfaces. There is, therefore, much work that needs to be carried out in terms of both the fundamentals of solid-binding kinetics of peptides and the effects of peptide primary and secondary structures on their recognition and binding to solid materials. Here we discuss the effects of constraints imposed on FliTrx-selected gold-binding peptide molecular structures upon their quantitative gold-binding affinity. We first selected two novel gold-binding peptide (AuBP) sequences using a FliTrx random peptide display library. These were, then, synthesized in two different forms: cyclic (c), reproducing the original FliTrx gold-binding sequence as displayed on bacterial cells, and linear (l) dodecapeptide gold-binding sequences. All four gold-binding peptides were then analyzed for their adsorption behavior using surface plasmon resonance spectroscopy. The peptides exhibit a range of binding affinities to and adsorption kinetics on gold surfaces, with the equilibrium constant, Keq, varying from 2.5x10(6) to 13.5x10(6) M(-1). Both circular dichroism and molecular mechanics/energy minimization studies reveal that each of the four peptides has various degrees of random coil and polyproline type II molecular conformations in solution. We found that AuBP1 retained its molecular conformation in both the c- and l-forms, and this is reflected in having similar adsorption behavior. On the other hand, the c- and l-forms of AuBP2 have different molecular structures, leading to differences in their gold-binding affinities.