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We have developed a rapid and efficient way of stretching DNA and denatured protein molecules for detection by fluorescence microscopy and atomic force microscopy (AFM). In the described method, a viscous drag created by a transient rotational flow stretches randomly coiled DNA molecules or denatured proteins. Stretching is achieved by dispensing a droplet of sample solution containing DNA or denatured protein on a MgCl(sub 2)-soaked mica surface. We present fluorescent images of straightened lambda DNA molecules and AFM images of stress-sheared, reduced von Willebrand factor as well as straightened lambda DNA. The described quick and reliable spin-stretching technique will find wide applications in the analysis of single biopolymer molecules.

We have developed a fluorescence-based method for mapping single or multiple protein-binding sites on straightened, large-size DNA molecules (> 5 kbp). In the described method, protein-DNA complexes were straightened and immobilized on a fiat surface using surface tension. A fraction of the immobilized complexes displayed a sharp DNA bend with two DNA segments extending from the apex. The presence of DNA-binding proteins at the apex was verified by atomic force microscopy. The position of protein binding relative to the ends of the DNA molecule was determined by measuring the length of two DNA segments using fluorescence microscopy. We demonstrate the potential of the fluorescence-based method to localize protein-binding sites on the DNA template and to evaluate relative binding affinity. The proposed protein-binding-site mapping technique is simple and easy to perform. Practical applications include screening for DNA-binding proteins and the localization of protein-binding sites on large segments of DNA.

The crystal structure of the nanoring subunit composed of the Escherichia coli DHFR dimer and a methotrexate dimerizer (MTX2-C9) containing a visible nine methylene linker and a protocol for the preparation of Chemically Self-Assembled Antibody Nanorings (CSANs) from the subunit with valencies similar to IgM's (decavalent) is described. The results have shown that anti-CD3CSANs is used for radionuclide, drug, or potentially oligonucleotide delivery to T-cells without the deleterious effects of activation observed for mAB.

The nonequilibrium synthesis of Cu2O nanoparticles is accomplished using an engineered derivative of the DNA-binding protein Tral in a room-temperature precursor electrolyte. The results show that a protein with no intrinsic inorganic synthesis activity can be endowed with the ability to control the formation of inorganic nanostructures under thermodynamically unfavorable conditions, reproducing a key feature of biological hard-tissue growth and assembly.

Chiral recognition of peptides on solid surfaces has been studied for a better understanding of their assembly mechanism toward its applications in stereochemistry and enantioselective catalysis. However, moving from small peptides such as dipeptides, understanding the chiral recognition of larger biomolecules such as oligopeptides or peptides with a larger sequence is challenging. Furthermore, their intrinsic mechanism for chiral recognition in liquid conditions was poorly investigated experimentally. Here, we used in/ex situ atomic force microscopy (AFM) to investigate the chiral recognition of self-assembled structures of l/d-type peptides on molybdenum disulfide (MoS 2 ). We chose single-layer MoS 2 with a triangular shape as a substrate for the self-assembly of peptides. The facet edges of MoS 2 were utilized as a landmark to identify the crystallographic orientation of their ordered structures. We found both peptide enantiomers formed nanowires on MoS 2 with a mirror symmetry according to the facet edges of MoS 2 . From in situ AFM measurements, we found a dimension of a unit cell in the self-assembled structure and proposed a model of lattice matching between peptides and MoS 2 lattice. The lattice matching for chiral recognition was further investigated by changing peptide sequences and surface lattice from MoS 2 to graphite. This work further deepened the understanding of biomolecular chiral recognition and will lead us to rationally design specific morphologies and conformations of chiral self-assembled structures of peptides with expected functions in the future.

Key to the development of functional bioinorganic soft interfaces is the predictive control over the micron-scale assembly structure and energetics of biomolecules at solid interfaces. While assembly of labile biomolecules, such as short peptides, at interfaces is a great deal affected by the shape of the molecule, biomolecular conformations are prompted by external solution conditions, involving temperature, pH, and salt concentration. In this light, one can expect that the environmental conformational selection of aqueous biomolecules could potentially allow for fine-tuning of the equilibrium assembly structure at interfaces, as well as, the binding strength and molecular mobility within these assemblies. Here, we demonstrate the energetic and structural tailoring of two-dimensional surface assemblies of graphite-binding dodecapeptides, through the thermal selection of aqueous peptide conformations. Our findings based on a scanning probe energetic analysis, supplemented by molecular dynamics modeling, show that peptide-graphite and peptide-peptide intermolecular interactions strongly depend on the thermally selected molecular conformation and that the extent of the conformational change is directly related to the observed assembled structure. Enabled by these results was the design of a peptide with predictable binding and assembled structure, thus, suggesting environmental preconditioning of peptides as a means for controlling self-assembling active bioinorganic interfaces for bioelectronic implementations such as biomolecular fuel cells and biosensors.

White spot lesions (WSL) and incipient caries on enamel surfaces are the earliest clinical outcomes for demineralization and caries. If left untreated, the caries can progress and may cause complex restorative procedures or even tooth extraction which destroys soft and hard tissue architecture as a consequence of connective tissue and bone loss. Current clinical practices are insufficient in treating dental caries. A long-standing practical challenge associated with demineralization related to dental diseases is incorporating a functional mineral microlayer which is fully integrated into the molecular structure of the tooth in repairing damaged enamel. This study demonstrates that small peptide domains derived from native protein amelogenin can be utilized to construct a mineral layer on damaged human enamel in vitro. Six groups were prepared to carry out remineralization on artificially created lesions on enamel: (1) no treatment, (2) Ca 2+ and PO 4 3- only, (3) 1100 ppm fluoride (F), (4) 20 000 ppm F, (5) 1100 ppm F and peptide, and (6) peptide alone. While the 1100 ppm F sample (indicative of common F content of toothpaste for homecare) did not deliver F to the thinly deposited mineral layer, high F test sample (indicative of clinical varnish treatment) formed mainly CaF 2 nanoparticles on the surface. Fluoride, however, was deposited in the presence of the peptide, which also formed a thin mineral layer which was partially crystallized as fluorapatite. Among the test groups, only the peptide-alone sample resulted in remineralization of fairly thick (10 μm) dense mineralized layer containing HAp mineral, resembling the structure of the healthy enamel. The newly formed mineralized layer exhibited integration with the underlying enamel as evident by cross-sectional imaging. The peptide-guided remineralization approach sets the foundation for future development of biomimetic products and treatments for dental health care.

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.

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.

Semiconductor nanocrystal quantum dots are utilized in numerous applications in nano- and biotechnology. In device applications, where several different material components are involved, quantum dots typically need to be assembled at explicit locations for enhanced functionality. Conventional approaches cannot meet these requirements where assembly of nanocrystals is usually material-nonspecific, thereby limiting the control of their spatial distribution. Here we demonstrate directed self-assembly of quantum dot emitters at material-specific locations in a color-conversion LED containing several material components including a metal, a dielectric, and a semiconductor. We achieve a spatially selective immobilization of quantum dot emitters by using the unique material selectivity characteristics provided by the engineered solid-binding peptides as smart linkers. Peptide-decorated quantum dots exhibited several orders of magnitude higher photoluminescence compared to the control groups, thus, potentially opening up novel ways to advance these photonic platforms in applications ranging from chemical to biodetection.

The assembly kinetics of colloidal semiconductor quantum dots (QDs) on solid inorganic surfaces is of fundamental importance for implementation of their solid-state devices. Herein an inorganic binding peptide, silica binding QBP1, was utilized for the self-assembly of nanocrystal quantum dots on silica surface as a smart molecular linker. The QD binding kinetics was studied comparatively in three different cases: first, QD adsorption with no functionalization of substrate or QD surface; second, QD adsorption on QBP1-modified surface; and, finally, adsorption of QBP1-functionalized QD on silica surface. The surface modification of QDs with QBP1 enabled 79.3-fold enhancement in QD binding affinity, while modification of a silica surface with QBP1 led to only 3.3-fold enhancement. The fluorescence microscopy images also supported a coherent assembly with correspondingly increased binding affinity. Decoration of QDs with inorganic peptides was shown to increase the amount of surface-bound QDs dramatically compared to the conventional methods. These results offer new opportunities for the assembly of QDs on solid surfaces for future device applications., (© 2011 American Chemical Society)

We use replica-exchange molecular dynamics (REMD) to interrogate molecular structures and properties of four engineered dodecapeptides (in solution, in the absence of a surface) that have been shown to bind to quartz with different propensities. We find that all of the strong-binding peptides feature some polyproline type II secondary structure, have less conformational freedom, and feature fewer intrapeptide hydrogen bonds compared with the weak binder. The regions of contiguous proline content in a given sequence appear to play a role in fostering some of these properties of the strong binders. For preliminary insights into quartz binding, we perform lattice-matching studies between a grid corresponding with the quartz (100) surface and the strong-binding peptide REMD structures. Our findings indicate a commonality among the putative contact residues, even for peptide structures with very different backbone conformations. Furthermore, interpeptide interactions in solution are studied. Our preliminary findings indicate that the strong-binder interpeptide contacts are dominated by weak, nonspecific hydrophobic interactions, while the weak-binding peptide shows more variable behavior due to the distribution of charged residues. In summary, the solution structures of peptides appear to be significant. We propose that these differences in their intra- and interpeptide interactions can influence their propensity to bind onto a solid substrate.

A number of clever recombinant methodologies have been developed that recapitulate the valencies of IgG's (bivalent) and IgA's (tetravalent). Although higher synthetic valencies have been achieved by conjugation of either monoclonal antibodies or single-chain antibodies to nanoparticles and liposomes, a method for the preparation of recombinant antibodies with valencies similar to IgM's (decavalent) but considerably less than what is generally found after antibody particle conjugation has yet to be devised. Recently, we have developed a methodology for the design of bivalent Chemically Self-Assembled Antibody Nanorings (CSANs). We now report the crystal structure of the nanoring subunit composed of the E. coli DHFR dimer and a methotrexate dimerizer (MTX2-C9) containing a visible nine methylene linker and a protocol for the preparation of CSANs from this subunit with valencies similar to IgM's, ranging from 8-10 single chain antibodies (scFvs). The multivalent CSANs were reversibly assembled from a fusion protein dihydrofolate reductase (DHFR)-DHFR-antiCD3 scFv containing a single glycine linker between the two DHFR scaffolding proteins. We also demonstrate that, similar to the parental bivalent anti-CD3 monoclonal antibody (mAB), anti-CD3 CSANs selectively bind to CD3+ leukemia cells and undergo rapid internalization through a caveolin-independent pathway that requires cholesterol, actin polymerization, and protein tyrosine kinase activation. While treatment with the monoclonal antibody leads to T-cell activation and nearly complete loss (i.e., 90%) of the surface displayed T-cell receptor (TCR), only 25-30% of the TCR down regulate and no significant T-cell proliferation is observed after treatment of peripheral blood mononuclear cells (PBMCs) with anti-CD3 CSANs. Consistent with the proliferation findings, 15-25% less CD25 (IL-2 receptor) was found on the surface of PBMCs treated with either the polyvalent or bivalent anti-CD3 CSANs, respectively, than on PBMCs treated with the parental mAB. Comparative experiments with F(ab')2 derived from the mAB confirm that the activation of the T-cells by the mAB is dependent on the Fc domain, and thus interactions of the PBMC T-cells with accessory cells, such as macrophages. Taken together, our results demonstrate that anti-CD3 CSANs with valencies ranging from 2 to 8 could be employed for radionuclide, drug, or potentially oligonucleotide delivery to T-cells without, as has been observed for other antibody conjugated nanoparticles, the deleterious effects of activation observed for mAB. Further the CSAN construct may be adapted for the preparation of other multivalent scFvs.

The fluorescence from conjugated polymer assembled onto lithographically fabricated gold nanoarrays using genetically engineered peptides as molecular linkers is studied. A 16-fold increase in the photoluminescence of the conjugated polymer is observed when assembled on the optimized nanostructures due to surface plasmon enhanced fluorescence. This is achieved using a water-soluble cationic conjugated polymer, poly[(9,9-bis(6'-((N,N,N-trimethylammonium)hexyl)-2,7-fluorene)-co-4,7-di-2-thienyl-2,1,3-benzothiadiazole] dibromide (PFDBT-N(+)), systematically tuning the vertical distance of PFDBT-N(+) from the gold nanopillar surface using solid-specific peptide linkers and horizontally optimizing the localized surface plasmon resonance by varying the geometric arrangements of the patterned metal nanoarrays. The diameter and tip-to-tip spacing of the nanopillars along with vertically tuning the distance of PFDBT-N(+) from the nanopillar affected the observed fluorescence enhancements. The collective optical properties of conjugated polymers combined with the photonic properties of nanoparticles provide a new means in the development of metal enhanced hybrid nanomaterials for biotechnology.

Understanding the mechanisms of biomineralization and the realization of biology-inspired inorganic materials formation largely depends on our ability to manipulate peptide/solid interfacial interactions. Material interfaces and biointerfaces are critical sites for bioinorganic synthesis, surface diffusion, and molecular recognition. Recently adapted biocombinatorial techniques permit the isolation of peptides recognizing inorganic solids that are used as molecular building blocks, for example, as synthesizers, linkers, and assemblers. Despite their ubiquitous utility in nanotechnology, biotechnology, and medicine, the fundamental mechanisms of molecular recognition of engineered peptides binding to inorganic surfaces remain largely unknown. To explore propensity rules connecting sequence, structure, and function that play key roles in peptide/solid interactions, we combine two different approaches: a statistical analysis that searches for highly enriched motifs among de novo designed peptides, and, atomistic simulations of three experimentally validated peptides. The two strong and one weak quartz-binding peptides were chosen for the simulations at the quartz (100) surface under aqueous conditions. Solution-based peptide structures were analyzed by circular dichroism measurements. Small and hydrophobic residues, such as Pro, play a key role at the interface by making close contact with the solid and hindering formation of intrapeptide hydrogen bonds. The high binding affinity of a peptide may be driven by a combination of favorable enthalpic and entropic effects, that is, a strong binder may possess a large number of possible binding configurations, many of which having relatively high binding energies. The results signify the role of the local molecular environment among the critical residues that participate in solid binding. The work herein describes molecular conformations inherent in material-specific peptides and provides fundamental insight into the atomistic understanding of peptide/solid interfaces.

With recent developments of nanoscale engineering in the physical and chemical sciences and advances in molecular biology, molecular biomimetics is combining genetic tools and evolutionary approaches with synthetic nanoscale constructs to create a new hybrid methodology: genetically designed peptide-based molecular materials. Following the fundamental principles of genome-based design, molecular recognition, and self-assembly in nature, we can now use recombinant DNA technologies to design single or multifunctional peptides and peptide-based molecular constructs that can interact with solids and synthetic systems. These solid-binding peptides have made significant impact as inorganic synthesizers, nanoparticle linkers, and molecular assemblers, or simply as molecular building blocks, in a wide range of fields from chemistry to materials science to medicine. As part of the programmatic theme, "Nanoscience: Challenges for the Future", the current developments, challenges, and future prospects of the field were presented during a symposium at the 237th ACS National Meeting held in March 2009. This Nano Focus article presents a synopsis of the work discussed there.

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 today's microelectronics, the fundamental observations of peptide-solid interactions here may well form the basis of peptide-based hybrid molecular technologies of the future.

Binding kinetics of platinum-, silica-, and gold-binding peptides were investigated using a modified surface plasmon resonance spectroscopy (SPR). Platinum binding septa-peptides, quartz-binding dodecapeptides, and gold-binding 14-aa peptides were originally selected using phage or cell surface display libraries using the mineral or pure forms of these materials. All of the peptides were synthesized singly to investigate their binding kinetics and to assess quantitatively the specific affinity of each to its material of selection. The peptides were also postselection engineered to contain multiple copies of the same original sequences to quantify the effects of repeating units. SPR spectroscopy, normally using gold surfaces, was modified to contain a thin film (a few nm thick) of the material of interest (silica or platinum) on gold to allow the quantitative study of the adsorption kinetics of specific solid-binding peptides. The SPR experiments, carried out at different concentrations, on all three materials substrates, resulted in Langmuir behavior that allowed the determination of the kinetic parameters, including adsorption, desorption, and equilibrium binding constants for each of the solids as well as free energy of adsorption. Furthermore, we also tested multiple repeats of the peptide sequences, specifically three repeats, to see if there is a general trend of increased binding with increased number of binding domains. There was no general trend in the binding strength of the peptides with the increase of the repeat units from one to three, possibly because of the conformational changes between the single and multiple repeat polypeptides. In all cases, however, the binding was strong enough to suggest that these inorganic binding peptides could potentially be used as specific molecular linkers to bind molecular entities to specific solid substrates due to their surface recognition characteristics.

We have demonstrated that nanostructures, and in particular nanorings incorporating a homodimeric enzyme, can be prepared by chemically induced self-assembly of dihydrofolate reductase (DHFR)-histidine triad nucleotide binding 1 (Hint1) fusion proteins. The dimensions of the nanorings were found by static light scattering and atomic force microscopy studies to be dependent on the length and composition of the peptide linking the fusion proteins, ranging in size from 10 to 70 nm in diameter and 64 to 740 kDa. The catalytic efficiency of the nanorings was found to be dependent on ring size, thus suggesting that the arrangement of supermolecular assemblies of enzymes may be used to control their catalytic parameters.

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.

Transmission electron microscopy was used to analyze the three-dimensional (3D) architecture of cuprous oxide electrochemically deposited through the pores of the hexagonally packed intermediate surface-layer protein from Deinococcus radiodurans SARK. Imaging at multiple tilt angles and averaging from five different samples allowed approximately 3 nm computed 3D reconstructions of the inorganic deposit and protein template. We show that the electrodeposition process used here was able to fully access the pore structure that penetrates the protein layer, allowing the fabrication of a polycrystalline nanoarray with 18 nm periodicity and lateral interconnectivity among the pores with 3-fold symmetry. At the resolution of the reconstruction, the 6-fold symmetry pores also appear filled but are not connected laterally to the rest of the deposit. These results show that electrochemical deposition can produce interconnected 3D structures at dimensions an order of magnitude smaller than the most advanced integrated circuits (IC), boding well for continued down-scaling of electrodeposition to meet the needs for future generations of IC device interconnects.

We report selection and characterization of hydroxyapatite-binding heptapeptides from a peptide-phage library and demonstrate the effects of two peptides, with different binding affinities and structural properties, on the mineralization of calcium phosphate mineral. In vitro mineralization studies carried out using one strong- and one weak-binding peptide, HABP1 and HABP2, respectively, revealed that the former exhibited a drastic outcome on mineralization kinetics and particle morphology. Strong-binding peptide yielded significantly larger crystals, as observed by electron microscopy, in comparison to those formed in the presence of a weak-binding peptide or in the negative control. Molecular structural studies carried out by circular dichroism revealed that HABP1 and HABP2 differed in their secondary structure and conformational stability. The results indicate that sequence, structure, and molecular stability strongly influence the mineralization activity of these peptides. The implication of the research is that the combinatorially selected short-sequence peptides may be used in the restoration or regeneration of hard tissues through their control over of the formation of calcium phosphate biominerals.

Recently, phage and cell-surface display libraries have been adapted for genetically selecting short peptides for a variety of inorganic materials. Despite the enormous number of inorganic-binding peptides reported and their bionanotechnological utility as synthesizers and molecular linkers, there is still a limited understanding of molecular mechanisms of peptide recognition of and binding to solid materials. As part of our goal of genetically designing these peptides, understanding the binding kinetics and thermodynamics, and using the peptides as molecular erectors, in this report we discuss molecular structural constraints imposed upon the quantitative binding characteristics of peptides with an affinity for inorganics. Specifically, we use a high-affinity seven amino acid Pt-binding sequence, PTSTGQA, as we reported in earlier studies and build two constructs: one is a Cys-Cys constrained "loop" sequence (CPTSTGQAC) that mimics the domain used in the pIII tail sequence of the phage library construction, and the second is the linear form, a septapeptide, without the loop. Both sequences were analyzed for their adsorption behavior on Pt thin films by surface plasmon resonance (SPR) spectroscopy and for their conformational properties by circular dichroism (CD). We find that the cyclic peptide of the integral Pt-binding sequence possesses single or 1:1 Langmuir adsorption behavior and displays equilibrium and adsorption rate constants that are significantly larger than those obtained for the linear form. Conversely, the linear form exhibits biexponential Langmuir isotherm behavior with slower and weaker binding. Furthermore, the structure of the cyclic version was found to adopt a random coil molecular conformation, whereas the linear version adopts a polyproline type II conformation in equilibrium with the random coil. The 2,2,2-trifluoroethanol titration experiments indicate that TFE has a different effect on the secondary structures of the linear and cyclic versions of the Pt binding sequence. We conclude that the presence of the Cys-Cys restraint affects both the conformation and binding behavior of the integral Pt-binding septapeptide sequence and that the presence or absence of constraints could be used to tune the adsorption and structural features of inorganic binding peptide sequences.

The adsorption kinetics of an engineered gold binding peptide on gold surface was studied by using both quartz crystal microbalance (QCM) and surface plasmon resonance (SPR) spectroscopy systems. The gold binding peptide was originally selected as a 14-amino acid sequence by cell surface display and then engineered to have a 3-repeat form (3R-GBP1) with improved binding characteristics. Both sets of adsorption data for 3R-GBP1 were fit to Langmuir models to extract kinetics and thermodynamics parameters. In SPR, the adsorption onto the surface shows a biexponential behavior and this is explained as the effect of bimodal surface topology of the polycrystalline gold substrate on 3R-GBP1 binding. Depending on the concentration of the peptide, a preferential adsorption on the surface takes place with different energy levels. The kinetic parameters (e.g., K(eq) approximately 10(7) M(-1)) and the binding energy (approximately -8.0 kcal/mol) are comparable to synthetic-based self-assembled monolayers. The results demonstrate the potential utilization of genetically engineered inorganic surface-specific peptides as molecular substrates due to their binding specificity, stability, and functionality in an aqueous-based environment.

We have developed a simple and robust method to fabricate nanoarrays of metals and metal oxides over macroscopic substrates using the crystalline surface layer (S-layer) protein of Deinococcus radiodurans as an electrodeposition mask. Substrates are coated by adsorption of the S-layer from a detergent-stabilized aqueous protein extract, producing insulating masks with 2-3 nm diameter solvent-accessible openings to the deposition substrate. The coating process can be controlled to achieve complete or fractional surface coverage. We demonstrate the general applicability of the technique by forming arrays of cuprous oxide (Cu(2)O), Ni, Pt, Pd, and Co exhibiting long-range order with the 18 nm hexagonal periodicity of the protein openings. This protein-based approach to electrochemical nanofabrication should permit the creation of a wide variety of two-dimensional inorganic structures.