Christopher R. So

Directed self‐immobilization of alkaline phosphatase on micro‐patterned substrates via genetically fused metal‐binding peptide

Current biotechnological applications such as biosensors, protein arrays, and microchips require oriented immobilization of enzymes. The characteristics of recognition, self‐assembly and ease of genetic manipulation make inorganic binding peptides an ideal molecular tool for site‐specific enzyme immobilization. Herein, we demonstrate the utilization of gold binding peptide (GBP1) as a molecular linker genetically fused to alkaline phosphatase (AP) and immobilized on gold substrate. Multiple tandem repeats (n = 5, 6, 7, 9) of gold binding peptide were fused to N‐terminus of AP (nGBP1‐AP) and the enzymes were expressed in E. coli cells. The binding and enzymatic activities of the bi‐functional fusion constructs were analyzed using quartz crystal microbalance spectroscopy and biochemical assays. Among the multiple‐repeat constructs, 5GBP1‐AP displayed the best bi‐functional activity and, therefore, was chosen for self‐immobilization studies. Adsorption and assembly properties of the fusion enzyme, 5GBP1‐AP, were studied via surface plasmon resonance spectroscopy and atomic force microscopy. We demonstrated self‐immobilization of the bi‐functional enzyme on micro‐patterned substrates where genetically linked 5GBP1‐AP displayed higher enzymatic activity per area compared to that of AP. Our results demonstrate the promising use of inorganic binding peptides as site‐specific molecular linkers for oriented enzyme immobilization with retained activity. Directed assembly of proteins on solids using genetically fused specific inorganic‐binding peptides has a potential utility in a wide range of biosensing and bioconversion processes. Biotechnol. Bioeng. 2009;103: 696–705.

Controlling Self-Assembly of Engineered Peptides on Graphite by Rational Mutation

Self-assembly of proteins on surfaces is utilized in many fields to integrate intricate biological structures and diverse functions with engineered materials. Controlling proteins at bio-solid interfaces relies on establishing key correlations between their primary sequences and resulting spatial organizations on substrates. Protein self-assembly, however, remains an engineering challenge. As a novel approach, we demonstrate here that short dodecapeptides selected by phage display are capable of self-assembly on graphite and form long-range-ordered biomolecular nanostructures. Using atomic force microscopy and contact angle studies, we identify three amino acid domains along the primary sequence that steer peptide ordering and lead to nanostructures with uniformly displayed residues. The peptides are further engineered via simple mutations to control fundamental interfacial processes, including initial binding, surface aggregation and growth kinetics, and intermolecular interactions. Tailoring short peptides via their primary sequence offers versatile control over molecular self-assembly, resulting in well-defined surface properties essential in building engineered, chemically rich, bio-solid interfaces.

Biofunctionalization of materials for implants using engineered peptides

Uncontrolled interactions between synthetic materials and human tissues are a major concern for implants and tissue engineering. The most successful approaches to circumvent this issue involve the modification of the implant or scaffold surfaces with various functional molecules, such as anti-fouling polymers or cell growth factors. To date, such techniques have relied on surface immobilization methods that are often applicable only to a limited range of materials and require the presence of specific functional groups, synthetic pathways or biologically hostile environments. In this study we have used peptide motifs that have been selected to bind to gold, platinum, glass and titanium to modify surfaces with poly(ethylene glycol) anti-fouling polymer and the integrin-binding RGD sequence. The peptides have several advantages over conventional molecular immobilization techniques; they require no biologically hostile environments to bind, are specific to their substrates and could be adapted to carry various active entities. We successfully imparted cell-resistant properties to gold and platinum surfaces using gold- and platinum-binding peptides, respectively, in conjunction with PEG. We also induced a several-fold increase in the number and spreading of fibroblast cells on glass and titanium surfaces using quartz and titanium-binding peptides in conjunction with the integrin ligand RGD. The results presented here indicate that control over the extent of cell-material interactions can be achieved by relatively simple and biocompatible surface modification procedures using inorganic binding peptides as linker molecules.

Molecular recognition and supramolecular self-assembly of a genetically engineered gold binding peptide on Au{111}.

The understanding of biomineralization and realization of biology-inspired materials technologies depends on understanding the nature of the chemical and physical interactions between proteins and biominerals or synthetically made inorganic materials. Recently, combinatorial genetic techniques permit the isolation of peptides recognizing specific inorganic materials that are used as molecular building blocks for novel applications. Little is known about the molecular structure of these peptides and the specific recognition mechanisms onto their counterpart inorganic surfaces. Here, we report high-resolution atomic force microscopy (AFM), molecular simulation (MS), and geometrical docking studies that detail the formation of an ordered supramolecular self-assembly of a genetically engineered gold binding peptide, 3rGBP(1) ([MHGKTQATSGTIQS](3)), correlating with the symmetry of the Au{111} surface lattice. Using simulated annealing molecular dynamics (SA/MD) studies based on nuclear magnetic resonance (NMR), we confirmed the intrinsic disorder of 3rGBP(1) and identified putative Au docking sites where surface-exposed side chains align with both the <110> and <211> Miller indices of the Au lattice. Our results provide fundamental insight for an atomistic understanding of peptide/solid interfaces and the intrinsic disorder that is inherent in some of these peptide sequences. Analogous to the well-established atomically controlled thin-film heterostructure formation on semiconductor substrates, the basis of todays microelectronics, the fundamental observations of peptide-solid interactions here may well form the basis of peptide-based hybrid molecular technologies of the future.

Adsorption, Diffusion, and Self‐Assembly of an Engineered Gold‐Binding Peptide on Au(111) Investigated by Atomic Force Microscopy

Go for the gold! The structural evolution of peptide binding and assembly on a Au(111) surface is dynamic and involves surface diffusion and multiple stages of molecular thin-film topology development (see schematic depiction and corresponding AFM images). The new fundamental observations may form the basis of peptide-based novel hybrid molecular technologies of the future.

Peptide-directed co-assembly of nanoprobes on multimaterial patterned solid surfaces

Biocombinatorially selected solid-binding peptides, through their unique material affinity and selectivity, are a promising platform for building up complex hierarchical assemblies of nanoscale materials and molecular probes, targeted to specific practical solid surfaces. Here, we demonstrate the material-specific characteristics of engineered gold-binding and silica-binding peptides through co-assembly onto micro- and nano-patterned gold surfaces on silica substrates. To build hierarchical nanostructures on patterned solid surfaces, we utilize peptides as molecular tools and monitor their behavior by either conjugating biotin to them for specific affinity to streptavidin-coated QDot nanoparticles or labelling them with small fluorescent labels. This biomimetic peptide-based approach could be used as an alternative to conventional chemical coupling and surface functionalization techniques with substantial advantages, allowing simultaneous assembly of two or more inorganic nano-entities and/or molecular probes onto patterned inorganic solid substrates. The results have significant implications in a wide range of potential applications, including controlled assembly of hybrid nanostructures in bionanophotonic and biosensing devices.

Controlling the Surface Chemistry of Graphite by Engineered Self-Assembled Peptides

The systematic control over surface chemistry is a long-standing challenge in biomedical and nanotechnological applications for graphitic materials. As a novel approach, we utilize graphite-binding dodecapeptides that self-assemble into dense domains to form monolayer-thick long-range-ordered films on graphite. Specifically, the peptides are rationally designed through their amino acid sequences to predictably display hydrophilic and hydrophobic characteristics while maintaining their self-assembly capabilities on the solid substrate. The peptides are observed to maintain a high tolerance for sequence modification, allowing control over surface chemistry via their amino acid sequence. Furthermore, through a single-step coassembly of two differently designed peptides, we predictably and precisely tune the wettability of the resulting functionalized graphite surfaces from 44° to 83°. The modular molecular structures and predictable behavior of short peptides demonstrated here give rise to a novel platform for functionalizing graphitic materials that offers numerous advantages, including noninvasive modification of the substrate, biocompatible processing in an aqueous environment, and simple fusion with other functional biological molecules.

Bioelectronic interfaces by spontaneously organized peptides on 2D atomic single layer materials.

Self-assembly of biological molecules on solid materials is central to the “bottom-up” approach to directly integrate biology with electronics. Inspired by biology, exquisite biomolecular nanoarchitectures have been formed on solid surfaces. We demonstrate that a combinatorially-selected dodecapeptide and its variants self-assemble into peptide nanowires on two-dimensional nanosheets, single-layer graphene and MoS2. The abrupt boundaries of nanowires create electronic junctions via spatial biomolecular doping of graphene and manifest themselves as a self-assembled electronic network. Furthermore, designed peptides form nanowires on single-layer MoS2 modifying both its electric conductivity and photoluminescence. The biomolecular doping of nanosheets defined by peptide nanostructures may represent the crucial first step in integrating biology with nano-electronics towards realizing fully self-assembled bionanoelectronic devices.

Electrical detection of biomolecular adsorption on sprayed graphene sheets.

The binding affinities of graphite-binding peptides to a graphite surface were electrically characterized using sprayed graphene field effect transistors (SGFETs) fabricated with solution exfoliated graphene. The binding affinities of these peptides were also characterized using atomic force microscopy (AFM) and mechanically exfoliated graphene field effect transistors (GFETs) to confirm the validity of the SGFET platform. Binding constants obtained via GFET and AFM were comparable with those observed using SGFETs. The sprayed graphene film serves as a scalable platform to study biomolecular adsorption to graphitic surfaces.

Imaging Active Surface Processes in Barnacle Adhesive Interfaces

Surface plasmon resonance imaging (SPRI) and voltammetry were used simultaneously to monitor Amphibalanus (=Balanus) amphitrite barnacles reattached and grown on gold-coated glass slides in artificial seawater. Upon reattachment, SPRI revealed rapid surface adsorption of material with a higher refractive index than seawater at the barnacle/gold interface. Over longer time periods, SPRI also revealed secretory activity around the perimeter of the barnacle along the seawater/gold interface extending many millimeters beyond the barnacle and varying in shape and region with time. Ex situ experiments using attenuated total reflectance infrared (ATR-IR) spectroscopy confirmed that reattachment of barnacles was accompanied by adsorption of protein to surfaces on similar time scales as those in the SPRI experiments. Barnacles were grown through multiple molting cycles. While the initial reattachment region remained largely unchanged, SPRI revealed the formation of sets of paired concentric rings having alternately darker/lighter appearance (corresponding to lower and higher refractive indices, respectively) at the barnacle/gold interface beneath the region of new growth. Ex situ experiments coupling the SPRI imaging with optical and FTIR microscopy revealed that the paired rings coincide with molt cycles, with the brighter rings associated with regions enriched in amide moieties. The brighter rings were located just beyond orifices of cement ducts, consistent with delivery of amide-rich chemistry from the ducts. The darker rings were associated with newly expanded cuticle. In situ voltammetry using the SPRI gold substrate as the working electrode revealed presence of redox active compounds (oxidation potential approx 0.2 V vs Ag/AgCl) after barnacles were reattached on surfaces. Redox activity persisted during the reattachment period. The results reveal surface adsorption processes coupled to the complex secretory and chemical activity under barnacles as they construct their adhesive interfaces.