Mustafa Gungormus

Regulation of in vitro calcium phosphate mineralization by combinatorially selected hydroxyapatite-binding peptides.

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.

Molecular biomimetics: GEPI‐based biological routes to technology

In nature, the viability of biological systems is sustained via specific interactions among the tens of thousands of proteins, the major building blocks of organisms from the simplest single‐celled to the most complex multicellular species. Biomolecule‐material interaction is accomplished with molecular specificity and efficiency leading to the formation of controlled structures and functions at all scales of dimensional hierarchy. Through evolution, Mother Nature developed molecular recognition by successive cycles of mutation and selection. Molecular specificity of probe‐target interactions, e.g., ligand‐receptor, antigen–antibody, is always based on specific peptide molecular recognition. Using biology as a guide, we can now understand, engineer, and control peptide‐material interactions and exploit them as a new design tool for novel materials and systems. We adapted the protocols of combinatorially designed peptide libraries, via both cell surface or phage display methods; using these we select short peptides with specificity to a variety of practical materials. These genetically engineered peptides for inorganics (GEPI) are then studied experimentally to establish their binding kinetics and surface stability. The bound peptide structure and conformations are interrogated both experimentally and via modeling, and self‐assembly characteristics are tested via atomic force microscopy. We further engineer the peptide binding and assembly characteristics using a computational biomimetics approach where bioinformatics based peptide‐sequence similarity analysis is developed to design higher generation function‐specific peptides. The molecular biomimetic approach opens up new avenues for the design and utilization of multifunctional molecular systems in a wide‐range of applications from tissue engineering, disease diagnostics, and therapeutics to various areas of nanotechnology where integration is required among inorganic, organic and biological materials. Here, we describe lessons from biology with examples of protein‐mediated functional biological materials, explain how novel peptides can be designed with specific affinity to inorganic solids using evolutionary engineering approaches, give examples of their potential utilizations in technology and medicine, and, finally, provide a summary of challenges and future prospects.

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.

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.

Cementomimetics—constructing a cementum-like biomineralized microlayer via amelogenin-derived peptides

Cementum is the outer-, mineralized-tissue covering the tooth root and an essential part of the system of periodontal tissue that anchors the tooth to the bone. Periodontal disease results from the destructive behavior of the host elicited by an infectious biofilm adhering to the tooth root and left untreated, may lead to tooth loss. We describe a novel protocol for identifying peptide sequences from native proteins with the potential to repair damaged dental tissues by controlling hydroxyapatite biomineralization. Using amelogenin as a case study and a bioinformatics scoring matrix, we identified regions within amelogenin that are shared with a set of hydroxyapatite-binding peptides (HABPs) previously selected by phage display. One 22-amino acid long peptide regions referred to as amelogenin-derived peptide 5 (ADP5) was shown to facilitate cell-free formation of a cementum-like hydroxyapatite mineral layer on demineralized human root dentin that, in turn, supported attachment of periodontal ligament cells in vitro. Our findings have several implications in peptide-assisted mineral formation that mimic biomineralization. By further elaborating the mechanism for protein control over the biomineral formed, we afford new insights into the evolution of protein–mineral interactions. By exploiting small peptide domains of native proteins, our understanding of structure–function relationships of biomineralizing proteins can be extended and these peptides can be utilized to engineer mineral formation. Finally, the cementomimetic layer formed by ADP5 has the potential clinical application to repair diseased root surfaces so as to promote the regeneration of periodontal tissues and thereby reduce the morbidity associated with tooth loss.

Peptide-mediated surface-immobilized quantum dot hybrid nanoassemblies with controlled photoluminescence

Combinatorially selected peptides and peptide–organic conjugates were used as linkers with controlled structural and organizational conformations to attach quantum dots (QDs) at addressable distances from a metal surface. This study demonstrates an approach towards nanophotonics by integrating inorganic, organic, and biological constructs to form hybrid nanoassemblies through template-directed self-assembly. Peptide–organic-linked QD arrays showed stronger fluorescence than peptide-linked QD arrays. We attribute this difference primarily to the increased number density of QDs on peptide–organic-linked QD arrays.

Ferro-microfluidic device for pathogen detection

In a previous work, we demonstrated that traveling wave excitations from integrated electrodes can continuously pump magnetic liquids within a microfluidic channel. The optimum excitation frequency of this pumping is strongly dependent on the hydrodynamic size of the magnetic nanoparticles, and the effect can be used to detect whenever a molecule or pathogen binds to the magnetic nanoparticles within a ferrofluid. Here, we demonstrate the bio-functionality and pathogen detection capability of a ferrofluid comprised of cobalt-ferrite-silica nanoparticles through the use of biotinylated genetically engineered peptides for inorganics (GEPs). These biotinylated GEPIs are specifically engineered to attach to the silica surface of the magnetic nanoparticles. Binding of streptavidin to the biotinylated GEPIs on the surface of the magnetic nanoparticles shifts the optimum pumping frequency by an amount that corresponds to the increase in the hydrodynamic size of the nanoparticle. The combination of GEPI-enhanced ferrofluids with integrated microfluidic devices finally enables the development of highly sensitive, portable and cheap pathogen sensor chips.

Nanoscale self-assembly mediated by genetically engineered gold binding polypeptide

We present a self-assembly method for guiding and positioning nano- and microscale objects onto a template mediated by a genetically engineered polypeptide. We have identified a polypeptide that can specifically recognize and bind to gold via cell surface display and biopanning. We demonstrate that this polypeptide can differentiate between various inorganic materials such as platinum, gold, and silicon dioxide. We have utilized this polypeptide to guide the assembly of nanoscale quantum dots or microscale gold spheres onto patterned microfabricated substrates. Our approach of using the material recognition polypeptides opens a new venue for bridging the organic and inorganic domains and guiding self-assembly of structures and devices from the bottom up.