Urartu Ozgur Safak Seker

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.

Material Binding Peptides for Nanotechnology

Remarkable progress has been made to date in the discovery of material binding peptides and their utilization in nanotechnology, which has brought new challenges and opportunities. Nowadays phage display is a versatile tool, important for the selection of ligands for proteins and peptides. This combinatorial approach has also been adapted over the past decade to select material-specific peptides. Screening and selection of such phage displayed material binding peptides has attracted great interest, in particular because of their use in nanotechnology. Phage display selected peptides are either synthesized independently or expressed on phage coat protein. Selected phage particles are subsequently utilized in the synthesis of nanoparticles, in the assembly of nanostructures on inorganic surfaces, and oriented protein immobilization as fusion partners of proteins. In this paper, we present an overview on the research conducted on this area. In this review we not only focus on the selection process, but also on molecular binding characterization and utilization of peptides as molecular linkers, molecular assemblers and material synthesizers.

A novel knowledge-based approach to design inorganic-binding peptides

MOTIVATION The discovery of solid-binding peptide sequences is accelerating along with their practical applications in biotechnology and materials sciences. A better understanding of the relationships between the peptide sequences and their binding affinities or specificities will enable further design of novel peptides with selected properties of interest both in engineering and medicine. RESULTS A bioinformatics approach was developed to classify peptides selected by in vivo techniques according to their inorganic solid-binding properties. Our approach performs all-against-all comparisons of experimentally selected peptides with short amino acid sequences that were categorized for their binding affinity and scores the alignments using sequence similarity scoring matrices. We generated novel scoring matrices that optimize the similarities within the strong-binding peptide sequences and the differences between the strong- and weak-binding peptide sequences. Using the scoring matrices thus generated, a given peptide is classified based on the sequence similarity to a set of experimentally selected peptides. We demonstrate the new approach by classifying experimentally characterized quartz-binding peptides and computationally designing new sequences with specific affinities. Experimental verifications of binding of these computationally designed peptides confirm our predictions with high accuracy. We further show that our approach is a general one and can be used to design new sequences that bind to a given inorganic solid with predictable and enhanced affinity.

Quantitative Affinity of Genetically Engineered Repeating Polypeptides to Inorganic Surfaces

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.

In vitro labeling of hydroxyapatite minerals by an engineered protein

Biological and biomimetic synthesis of inorganics have been a major focus in hard tissue engineering as well as in green processing of advanced materials. Among the minerals formed by organisms, calcium phosphate mineralization is studied extensively to understand the formation of mineral‐rich tissues. Herein, we report an engineered fusion protein that not only targets calcium phosphate minerals but also allows monitoring of biomineralization. To produce the bi‐functional fusion protein, nucleotide sequence encoding combinatorially selected hydroxyapatite‐binding peptides (HABP) was genetically linked to the 3′ end of the open reading frame of green fluorescence protein (GFPuv) and successfully expressed in Escherichia coli. The fluorescence and binding activities of the bi‐functional proteins were characterized by, respectively, using fluorescence microscopy and quartz crystal microbalance spectroscopy. The utility of GFPuv‐HABP fusion protein was assessed for both time‐wise monitoring of mineralization and the visualization of the mineralized tissues. We used an alkaline phosphatase‐based reaction to control phosphate release, thereby mimicking biological processes, to monitor calcium phosphate mineralization. The increase in mineral amount was observed using the fusion protein at different time points. GFPuv‐HABP1 was also used for efficient fluorescence labeling of mineralized regions on the extracted human incisors. Our results demonstrate a simple and versatile application of inorganic‐binding peptides conjugated with bioluminescence proteins as bi‐functional bioimaging molecular probes that target mineralization, and which can be employed to a wide range of biomimetic processing and cell‐free tissue engineering. Bioeng. 2011; 108:1021–1030.

Spatially Selective Assembly of Quantum Dot Light Emitters in an LED Using Engineered Peptides

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.

Peptide-mediated constructs of quantum dot nanocomposites for enzymatic control of nonradiative energy transfer.

A bottom-up approach for constructing colloidal semiconductor quantum dot (QDot) nanocomposites that facilitate nonradiative Förster-type resonance energy transfer (FRET) using polyelectrolyte peptides was proposed and realized. The electrostatic interaction of these polypeptides with altering chain lengths was probed for thermodynamic, structural, and morphological aspects. The resulting nanocomposite film was successfully cut with the protease by digesting the biomimetic peptide layer upon which the QDot assembly was constructed. The ability to control photoluminescence decay lifetime was demonstrated by proteolytic enzyme activity, opening up new possibilities for biosensor applications.

Synthetic Biogenesis of Bacterial Amyloid Nanomaterials with Tunable Inorganic-Organic Interfaces and Electrical Conductivity.

Amyloids are highly ordered, hierarchal protein nanoassemblies. Functional amyloids in bacterial biofilms, such as Escherichia coli curli fibers, are formed by the polymerization of monomeric proteins secreted into the extracellular space. Curli is synthesized by living cells, is primarily composed of the major curlin subunit CsgA, and forms biological nanofibers with high aspect ratios. Here, we explore the application of curli fibers for nanotechnology by engineering curli to mediate tunable biological interfaces with inorganic materials and to controllably form gold nanoparticles and gold nanowires. Specifically, we used cell-synthesized curli fibers as templates for nucleating and growing gold nanoparticles and showed that nanoparticle size could be modulated as a function of curli fiber gold-binding affinity. Furthermore, we demonstrated that gold nanoparticles can be preseeded onto curli fibers and followed by gold enhancement to form nanowires. Using these two approaches, we created artificial cellular systems that integrate inorganic-organic materials to achieve tunable electrical conductivity. We envision that cell-synthesized amyloid nanofibers will be useful for interfacing abiotic and biotic systems to create living functional materials..

Assembly Kinetics of Nanocrystals via Peptide Hybridization

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.

Bio-nanohybrids of quantum dots and photoproteins facilitating strong nonradiative energy transfer

Utilization of light is crucial for the life cycle of many organisms. Also, many organisms can create light by utilizing chemical energy emerged from biochemical reactions. Being the most important structural units of the organisms, proteins play a vital role in the formation of light in the form of bioluminescence. Such photoproteins have been isolated and identified for a long time; the exact mechanism of their bioluminescence is well established. Here we show a biomimetic approach to build a photoprotein based excitonic nanoassembly model system using colloidal quantum dots (QDs) for a new bioluminescent couple to be utilized in biotechnological and photonic applications. We concentrated on the formation mechanism of nanohybrids using a kinetic and thermodynamic approach. Finally we propose a biosensing scheme with an ON/OFF switch using the QD-GFP hybrid. The QD-GFP hybrid system promises strong exciton-exciton coupling between the protein and the quantum dot at a high efficiency level, possessing enhanced capabilities of light harvesting, which may bring new technological opportunities to mimic biophotonic events.