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Review

Molecular biomimetics: Utilizing natures molecular ways inpractical engineering q

Candan Tamerler a,b, Mehmet Sarikaya a,c,*

a Materials Science and Engineering, University of Washington, Seattle, WA 98195, USAb Molecular Biology and Genetic, Istanbul Technical University, Maslak, 34 469 Istanbul, Turkey

c Chemical Engineering, University of Washington, Seattle, WA 98195, USA

Received 9 June 2006; received in revised form 22 October 2006; accepted 24 October 2006

Abstract

In nature, proteins are the machinery that accomplish many functions through their specific recognition and interactions in bio-logical systems from single-celled to multicellular organisms. Biomoleculematerial interaction is accomplished via molecular specific-ity, leading to the formation of controlled structures and functions at all scales of dimensional hierarchy. Through evolution,molecular recognition and, consequently, functions developed through successive cycles of mutation and selection. Using biologyas a guide, we can now understand, engineer and control peptidematerial interactions and exploit these to tailor novel materialsand systems for practical applications. We adapted combinatorial biology protocols to display peptide libraries, either on the cell sur-face or on phages, to select short peptides specific to a variety of practical materials systems. Following the selection step, we deter-mined the kinetics and stability of peptide binding experimentally to understand the bound peptide structure via modeling and itsassembly via atomic force microscopy. The peptides were further engineered to have multiple repeats or their amino acid sequencesvaried to tailor their function. Both nanoparticles and flat inorganic substrates containing multimaterials patterned at the nano- and

microscales were used for self-directed immobilization of molecular constructs. The molecular biomimetic approach opens up newavenues for the design and utilization of multifunctional molecular systems with wide ranging applications, from tissue engineering,drug delivery and biosensors, to nanotechnology and bioremediation. Here we give examples of protein-mediated functional materialsin biology, peptide selection and engineering with affinity to inorganics, demonstrate potential utilizations in materials science, engi-neering and medicine, and describe future prospects. 2006 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Keywords: Molecular biomimetics; Inorganic-binding peptides; Multifunctional proteins; Hybrid materials and systems; Bionanotechnology

Contents

1. Introduction lessons from nature and molecular biomimetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2902. Genetically engineering proteins for inorganics (GEPI) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2903. Post-selection engineering. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2944. Current multidisciplinary applications of engineered polypeptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2955. Future prospects and potentials of molecular biomimetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 297

1742-7061/$ - see front matter 2006 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

doi:10.1016/j.actbio.2006.10.009

q Research presented at the TMS 2006 Biological Materials Science Symposium.* Corresponding author. Address: Materials Science and Engineering, University of Washington, Seattle, WA 98195, USA. Tel.: +1 206 543 0724;

fax: +1 206 543 3100.E-mail address: [email protected] (M. Sarikaya).

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Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 298References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 298

1. Introduction lessons from nature and molecular

biomimetics

Nature has evolved mechanisms of simplicity and ele-gance to synthesize soft and hard tissues exhibiting remark-able functional properties [14]. Nature achieves these featsof engineering by making use of molecular building blocksand by controlling materials assembly in a hierarchicalmanner from the nano- to the macroscale [57]. With agrowing understanding of the processes involved camethe realization that biological principles may have applica-tions for solving problems in human-made systems. Thereis indeed a rich and long history of gaining inspiration fromnatures biological structures to design practical materials

and systems. Biomimeticists have traditionally focused onemulating or duplicating biosystems using mostly syntheticcomponents and conventional approaches [13]. By merg-ing recent advances in molecular biology [8,9] with state-of-the-art engineering and physical sciences [1012], thenew goal in the emerging field of molecular biomimeticsis to shift biomimetic materials science away from imitatingto engineering materials as nature does to perform artificialfunctions from the molecular scale up [7]. The new researchis focused on combining proven biomolecular tools withsynthetic nanoscale constructs to make molecular biomi-metics a full-fledged methodology.

Nature provides abundant examples of multifunctionalmaterials, devices and systems that scientists could investi-gate to understand the bases of synthesis, formation andfunction, and engineers could then emulate their practicalutility in everyday applications. In Fig. 1, we present thefour examples from our previous research where proteinscontrol engineered material systems formation in whichboth components, proteins and inorganics, are essentialfor their synthesis, assembly and function [1317]. Forexample, in magnetotactic bacteria (Fig. 1A), a biocompassis made of aligned magnetosomes that are composed ofprotein-based membrane compartments that controlFe2+, Fe3+ and O2 ion transport, synthesis, nucleation

and morphogenesis of the magnetite (Fe3O4) nanoparticles.Our second example is from an Antartic sponge; althoughresiding 200 m under the ocean, Rosella racovitzea has asymbiotic relation with green algae that live within its bar-rel-shaped body [13]. This is possible because of the silica-based spicules that collect and transmit light effectivelyacross the outer wall of the sponge (Fig. 1B). Both the spic-ular tip (a lens) and the shaft (optical fiber) are molecularcomposites of silica and bound proteins that provide thestructural, architectural and functional properties to thespicular system. The third is a classic biomimetic example:mother-of-pearl, the natural armor of mollusks shells. The

interior of the shell is constituted of nacre, a layered and

segmented hybrid composite of aragonite (orthorhombicCaCO3) and a biopolymer mixture, i.e. nanostructurallyintegrated proteins and polysaccharides (e.g. chitin) witha 95/5 inorganic/organic volume ratio (Fig. 1C). In spiteof what appears to be rudimentary components, both thearchitecture of the soft and hard phases, i.e. the bricksand mortar, and their chemical and mechanical couplingresult in this unique biocomposite with the highest specifictoughness and fracture strength of all known ceramic-based materials [14,15]. The fourth example is enamel; itis the crown of the tooth, the hardest material in the body,providing protective cover to the dental tissues of mamma-lians [16]. On the underneath, the enamel is integrated todentin, a softer and, therefore, tougher bone-like tissue that

provides an energy-absorbing cushion during cutting andchewing. The unique woven architecture of enamel pro-vides the essential network resistance to the mixed stressesduring mastication, thereby preventing premature fractureor failure (Fig. 1D).

The examples above and countless others have uniquearchitecture and functions, providing biodesign lessons toengineers and technologists. In addition to an inorganicmaterial, each biosystem involves many proteins that maycome into play spatially and temporally in a complex bio-scheme during the transport of species, synthesis, fabrica-tion, system integration and networking. Understanding

the roles of proteins during biofabrication would providemeans to develop protocols for regeneration or emulationof these tissues, as well as to develop novel hybrid materialsand systems with unique physical properties [17]. Forexample, in enamel, more than 40 different proteins areknown to take part in its formation; and in nacre, perhaps1020 different ones do the same. The complexity of under-standing the biochemical and biophysical nature of each ofthe proteins, their multifaceted interactions and their rolesin the biofabrication and functional performance of the tis-sue or organ is enormous, and requires a gargantuan taskfor immediate practical utilization in materials science.While this major goal is being undertaken in proteomics

and genomics, simple polypeptides with specific functionscould be incorporated into mainstream materials scienceand engineering in more practical ways. Molecular biomi-metics, through genetic selection of inorganic-binding pep-tides and their tailoring through post-selection engineeringwould be one way to achieve the immediate utilization ofnatures molecular ways to make practical materials withnovel applications [17].

2. Genetically engineering proteins for inorganics (GEPI)

Proteins offer three unique advantages in developing

future materials and systems: molecular recognition,

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self-assembly and genetic manipulation [17]. These con-cepts are depicted in Fig. 2 with examples from our currentresearch. Although the fundamental mechanism of the rec-ognition of a material surface by a short peptide is far fromclear, quantitative binding experiments and modeling givesome clues of how this might be possible. For example,as shown in Fig. 2A, a molecular dynamics model of con-straint platinum-binding septapeptides (CPTSTGQAC, Csproviding the constrained conformation to the peptidethrough disulfide bridging) reveals that this GEPI hassub-nanometer-scale protrusions (called polypods) thatmatch with the ideal metal surface, possibly interactingvia the polar groups [18]. It is conceivable, then, thatmolecular recognition leads to nucleation, growth andmorphogenesis of inorganics under favorable synthesisconditions, and crystal-specific display of peptides. Oncea peptide recognizes a material, it could also further self-arrange on the surface to form supramolecular architec-

tures. One such example is shown in Fig. 2B, where a

three-repeat gold-binding peptide forms a self-assembledmolecular organization with sixfold symmetry on the sur-face of Au(111) [17].

The third advantage is that the new approach is a gen-ome-based manufacturing technology, i.e. the proteinsand peptides coded by the genes (of bacteria, phage, andyeast) provide means to genetically modify them and,therefore, precisely engineer their practical functions. Here,we present the plasmid presentation of five repeats of aGBP being genetically fused to alkaline phosphatase to cre-ate a bi-functional molecule [19]. Developing under thisframework would provide a multidisciplinary platformtowards realizing hybrid building blocks in which the poly-peptides are tailored genetically while the synthetic compo-nent is designed for its specific chemical and physicalfunctions for a wide range of applications from materialsscience to medicine.

Combinatorial biology-based selection techniques have

been a major tool for a myriad of biotechnological

Fig. 1. Examples of functional biological materials systems: (A) Magnetotactic bacteria, e.g. Aquaspirillum magnetotacticum, have aligned magnetite cubo-octahedral-shaped particles that are ordered single crystals. (B) The spicules of Rosella racovitzea have star-shaped tips that act as gatherers of light, whichis pumped through the silica-based biological optical fiber to the interior of the sponge. (C) Nacre, mother-of-pearl, is a segmented laminated composite ofcalcium carbonate and biomolecules with excellent mechanical properties that provide an armor to mollusks such as the red abalone, Haliotis rufescens,shown here. (D) Mammalian enamel, such as from the mouse, is $100% hydroxyapatite that has a hierarchically ordered woven structure to enablemastication under complex mechanical stresses (the right is an SEM image of the fractured surface and the left is a schematics of the woven architecture)[17].

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applications, including characterization of receptor-anti-body binding sites, the study of proteinligand interac-

tions, and the isolation and directed evolution of enzymesand peptides for improved catalysis or altered bindingcharacteristics [20]. Among in vivo combinatorial selectiontechniques, phage and cell surface display methods havebeen used extensively. In phage and cell surface displaytechniques (PD and CSD, respectively), random sequencesof amino acids, encoded within a phage genome or on aplasmid, are exposed to the desired environment withinthe context of a protein that naturally localizes on the sur-face of the virus or cell, respectively. Outer membrane pro-teins, lipoproteins, fimbria and flagellar proteins have allbeen used for bacterial cell surface display, while the coatproteins of bacteriophages M13, fd and f1 have beenexploited to expose random peptides on virus surfaces[2023]. A common feature of these systems is that thegenetic information encoding the displayed molecule isphysically linked to the product by the displayingorganism.

We [7,17,24,25] and others [2629] have adapted combi-natorial biology technologies and selected GEPIs for met-als, oxides, minerals and semiconductors. In our group,using pentavalent M13 phage display (Fig. 3A) and multi-valent FliTrX bacterial flagellar display (Fig. 3D), we haveso far identified more than 1000 peptides binding to a vari-ety of inorganics. Here, randomized oligonucleotides, dis-

played on the coat proteins of bacteriphages or on the

cell surface, are inserted into either phage genome or plas-mids, respectively (Fig. 3B). The molecular library is then

exposed to the material of interest. Next, a chemical-basedelution protocol is carried out for the selection of the tightbinders followed by several washing and elution cycles(Fig. 3C). Once the tight binders are eluted from the sur-faces, their DNA is extracted and sequenced. Some exam-ples of the sequences identified by our group, includingnoble metals (Pt, Pd, Ag, Au), metal oxides (ZnO, Al2O3,SiO2), semiconductors (GaN, Cu2O, TiO2, ITO, sulfides,selenides) and other functional materials (hydroxyapatite,mica, graphite, calcite), are given in Fig. 4 [7,17,24,25].

Usually there are up to 50 or more peptides selected forany given inorganic material with various degrees of bind-ing strengths. These peptides can be referred as the first setof selected peptide sequences from an initial randomlibrary screening (Fig. 5). Although a convergence towardsa strong consensus in the amino acid residues of theselected peptides (high affinity and high specificity) is pos-sible in proteinprotein interactions, in our experience thismay be difficult in identifying a material-specific motif withdesirable characteristics. In natural evolution, progenywith improved features are obtained following recurringcycles of mutations and selection. Similarly, the informa-tion obtained from screening an initial library can be usedfor the construction of a second-generation combinatoriallibrary in which specific amino acids contributing to ligand

binding are held invariant while neighboring residues are

Fig. 2. The demonstration of the three pillars of molecular biomimetics molecular recognition, self-assembly and genetic manipulation: (A) Recognitionof platinum, a noble metal, by a phage-display selected Pt-binding septapeptide that forms polypod molecular architecture. (B) Genetically designed three-repeat gold-binding peptide that recognizes the Au(111) surface and assembles into a two-dimensional ordered supramolecular structure. (C) Plasmidconstruct used to genetically fuse five-repeat gold-binding peptide to an enzyme, alkaline phosphatase, resulting in a bi-functional molecule.



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Fig. 3. A schematic illustration of the combinatorial display protocols adapted from molecular biology for selecting material-specific peptides: (A) M13phage-display library. (B) Basic steps in preparing a random phage or flagella library. (C) Biopanning procedure using random libraries. (D) FliTrXflagella library.

Fig. 4. Molecular toolbox: examples of inorganic-binding peptides selected by our group via phage, cell surface or flagella display.



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randomized (Fig. 5) [17]. These libraries could be subjectedto a new screen to isolate higher affinity binders and/orsequences exhibiting a high degree of specificity for onematerial without interacting with other closely relatedcompounds.

As an alternative to increased binding affinity, one coulduse molecular biology approaches to create multiple repeatsof a binding motif, thereby generating a set of binders

exhibiting a tailored Kd for a particular material. Throughthis strategy we have engineered gold-binding peptide(GBP) [24]. In GBP, the second generation library was con-structed by maintaining a known gold-binding sequence(KTQATS) as well as a motif encountered in several gold-binding peptides (SKTS), randomizing intervening aminoacids and multimerizing these sequences. Another exampleis ArgXXArg (where X is any amino acid), where thetetrapeptide is implicated in metal oxide binding, withArgArg and ArgLys pairs allowing for discriminationbetween Cu2O and ZnO [25]. To understand the core aminoacids crucial to the binding process, one could utilize an ala-nine scan using site-directed mutagenesis, where each of theamino acid residues in the selected sequences is replaced byalanine one by one. For directed evolution of inorganic-binding polypeptides, one can keep the core amino acidsbut change the noncontributing ones with the residues toimprove both affinity and specificity. Starting from thefirst-generation GEPIs, the binding affinity could beassessed, initially qualitatively, using immunofluorescencemicroscopy. Then quantitative binding and kinetics canbe obtained using techniques such as quartz crystal micro-balance and surface plasmon resonance spectroscopy [3032]. The assembled molecular structures can also bevisualized using atomic force microscopy (Fig. 2B). Fur-

thermore, peptide conformational structures can be mod-

eled by molecular dynamics (Fig. 2A) or quantifiedexperimentally using nuclear magnetic resonance spectros-copy [17]. Although many sequences specific to differentinorganics have been identified by many groups [2629]up to now via application of display technologies, there isstill a limited number of quantitative binding characteriza-tions providing the essential data for the evaluation of poly-peptide-inorganic surface affinity and selectivity [3032].

Clearly, this information is essential in the robust designand realization of protein/inorganic hybrid materials.

3. Post-selection engineering

As ourunderstanding of inorganic binding grows througha synergistic combination of experimental and computa-tional approaches, one would expect to identify particulararrangements of residues necessary for binding in a directedmanner. This knowledge could be combined with bioinfor-matics approaches to computationally design GEPIs thathave tailored binding and functional properties (Fig. 5).For instance, we are developing a novel bioinformatics pro-tocol to identify sequence patterns that confer inorganic-binding specificity. Briefly, when one compares all the bind-ers isolated using PD or CSD for a given material with thosefor different materials, it is possible to assign sequence simi-larity scores using genetic algorithm procedures. In essence,we can create a BLOSUM-like matrix (typically used forcomparing the protein sequences of different organisms),which is optimized so that peptides with similar bindingproperties for a particular material have a very high scoreand those with dissimilar binding have a low score. Onecan then generate random sequences and perform similaritycomparisons to known binders, with the assumption that

peptides that have a significant match to a known binder

Fig. 5. A schematic illustrating the genetic routes to tailor and express second, and higher, generation inorganic-binding peptides for improvedfunctionality.



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are likely to bind with high affinity to that surface and com-bine results with the de novo structure prediction strategies.The resulting sequences would be experimentally tested,allowing fine-tuning of in silico predictions [33].

The genetic engineering-based approaches, e.g. recombi-nant DNA technology, can be utilized in many different

ways. For example, tuned-up GEPIs can be developedfor the desired affinity and specificity by computationaldesign and post-selection engineering following the combi-natorial selection (Fig. 5). Also, desired peptides can alsobe generated in an in vivo environment. The engineeredpeptides can be expressed in Escherichia coli, or otherhosts, with additional modifications such as introductionof cleavable hexahistidine tails to simplify fusion peptide

purification via Ni-NTA affinity chromatography. TheGEPIs can then be produced in desired amounts followingthe removal of purification tags to be utilized as tools formany applications (Fig. 5).

4. Current multidisciplinary applications of engineered

polypeptides

Controlled binding and assembly of proteins on inor-ganic substrates are at the core of bionanotechnologyand biological materials engineering [3335]. GEPI pro-vides the molecular means to anchor, couple, brace, displayand assemble functional molecules, nanoparticles andstructures [17]. The examples in Fig. 6 provide a summary

Fig. 6. Some examples of diverse applications of GEPI: (A) Morphogenesis of inorganic nanoparticles using a GEPI; flat gold particles form in thepresence of GBP1 during synthesis. (B) Immobilization of target protein (FE, ferritin) using biotinylated GBP1 that assembles on Au(111). (C)Competitive assembly of Pt-binding phage mutant on micropatterned platinum compared to surface silica (substrate is Si-wafer). (D) Cell sorting usingmutant cells with cuprous oxide-binding peptides displayed on their flagella. (E) Formation of silica gel using tetraethylorthosilicate and quartz-binding

phage mutant. (F) Directed immobilization of GBP1-AP construct on a gold substrate with retained phosphotase activity.



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of potential uses from our current work. Here the engi-neered peptides are used as molecular manipulators for awide range of applications. The peptides are made avail-able from our molecular toolbox, a protein data bank con-taining fully characterized GEPIs. Furthermore, geneticfusion of GEPIs into functional proteins, for example,

enables them to be used as more practical ligands for manybio- and bionanotechnological applications by directly uti-lizing multifunctional protein-based constructs.

Similar to morphogenesis in hard tissues (such as mol-lusk shells, bones, spicules and dental tissues) providedby inorganic-binding proteins, GEPIs could be used tocontrol the geometrical shapes and sizes of inorganic nano-particles (Fig. 6A). Here, we demonstrate that GBP affectsthe Au particle morphology through controlling crystalgrowth. Nanogold (monosize, 120 A diameter) particlescan be formed at ambient conditions using the well-knownFaradays technique by reducing AuCl3 by Na3C6H5O7 (orother reducing agents) [7]. Reducing the gold concentration

and temperature allows particle formation at a slower rate,giving the protein time to interact with surfaces during thegrowth and providing conditions to examine the effect ofgold binding during colloidal gold formation. We havetested more than 50 mutants in our study for their influenceon the rate of crystallization of nanogold particles, and oursequence analysis shows that two separate mutants thataccelerated the crystal growth also changed the particlemorphology from cubo-octahedral (the usual shape ofthe gold particles under equilibrium growth conditions)to flat, triangular or pseudo-hexagonal particles [24]. Thisnew observation is interesting in demonstrating the effect

of gold-binding peptides in crystal growth rather than tra-ditionally-assumed templating effect.

Target DNA and functional proteins adsorbed specifi-cally onto probe substrates are used to build microarrayssuitable for modern functional genomics, pharmogeneticsand proteomics [3335]. Gold-binding polypeptides,selected by cell surface display, are one of the first examplesof engineered polypeptides for inorganic surfaces. Theywere screened via random peptide libraries expressed onthe outer surface ofE. colias part of the maltodextrin porin,LamB protein. As we have discussed here and elsewhere,among the identified peptides, GBP1 (MHGKTQATSG-TIQS) has been well characterized by our group. InFig. 6B, a biotinylated form of GBP is utilized as a molecu-lar erector for ferritin attachment to the gold surface. Strep-tavidin linkage in ferritin provides a specific linkage forbiotin, and consequently ferritin adsorption on the gold sur-face is achieved by GBP binding on the surface [17].

The utilization of the ability of GEPI to selectively bindto a specific inorganic would allow development of newplatforms for virus, phage and cell sorting using micro-pat-terned substrates (Fig. 6C and D, respectively). In Fig. 6C,we observe the phage sorting on a platinum-patterned silicasurface through a specific platinum-binding peptide dis-played on M13 phage. To prepare the substrate, we used

an electrochemical approach in which platinum macrodots

were printed on a silica (glass) substrate [36]. Relatively flatand circular regions of nominally 50 lm diameter contain-ing metallic platinum regions were verified via scanningelectron microscopy (SEM) and the chemical distributionof Si, O and Pt elemental maps were obtained using energydispersive X-ray spectroscopy (not shown here). A fluores-

cently tagged phage mutant displaying the specific plati-num-binding peptide was exposed to the patternedsubstrate in a buffer solution, resulting in self-directedphage binding onto the Pt dots preferably over the silicaregions [36]. Similarly, in Fig. 6D, selective adsorption offluorescently labeled E. coli can be sorted by its bindingto the cuprous oxide regions rather than on cupric hydrox-ide. Here, the cell flagella express a specific dodecapeptidethat binds to cuprous oxide (Cu2O) with high affinity andchemically distinguishes it from Cu(OH)2 [25].

Our next example uses a mutant phage incorporating aninorganic binder. Here the GEPI is utilized for material-spe-cific synthesis and the three-dimensional construction of the

resultant material while being an integral component of it.A silica-based composite material is developed consistingof a silica-specific phage mutant that forms a three-dimen-sional network with silica as the filler (Fig. 6E). In nature,biosilica exhibits diverse shapes and structures. The pro-teins directing silica synthesis in biological systems havebeen studied extensively. For example, silaffins, extractedfrom the cell walls of a diatom, Cylindrotheca, or silicatein,extracted from the spounge spicules ofTerhya aurantia, arewell known examples [37,38]. Following natures examples,in our research we identified quartz-binding peptides usinga dodecapeptide phage display library selected using a sin-

gle-crystal substrate. Here, we used the strong quartz-bind-ing peptide (DS 202: RLNPPSQMDPPF) displayed onphage to test their effect on silica synthesis. Samples (con-taining 1013 pfu) were incubated in freshly prepared tetram-ethylorthosilicate (TMOS) solution for 34 min at roomtemperature. We next centrifuged the solution and, afterdiscarding the supernant, removed the precipitated samplesusing a syringe. The samples were either freeze-dried or air-dried before testing for the presence of silica. The micro-structure and the elemental composition of the silica formedwere examined using SEM and transmission electronmicroscopy, and energy-dispersive X-ray spectroscopy(not shown). The results demonstrate that the selective pep-tides can be utilized for the biofabrication of the inorganicmaterial in an aqueous environment [39].

Our final example concerns with the use of inorganic-binding peptides for self-oriented immobilization ofenzymes. Although many methods have been used toimmobilize enzymes, there has always been a compromisebetween trying to maintain the high activity of the enzymeand having the advantages of immobilization. The existingmolecular systems that have been the hallmark for self-assembly in chemistry are mostly thiol- and silane-based.Although these linkers have been used successfully so far,their utility can be limited because of their non-specific

interactions. The proteins immobilized through these



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linkers might be positioned on the surface in random orien-tations, resulting in a loss of their biological activity. Asdemonstrated in Fig. 6F, the inorganic-binding peptidescan be utilized as potential linkers, as not only is each spe-cific to an inorganic surface, but they also allow geneticconjugation to create bi- or multifunctional constructs.

The example here uses a five-repeat GBP genetically fusedto an enzyme, alkaline phosphatase (AP). The resulting bi-functional enzyme is then self-immobilized, homoge-neously covering the gold substrate (Fig. 6F) [19]. TheAP is an essential hydrolase enzyme for regulating (pre-venting or enhancing) biomineralization of hydroxyapatitevia control of the extracellular phosphate concentration bycatalyzing, for example, pyrophosphate degradation. Here,the bi-functional hybrid molecular construct keeps both itsenzymatic function and its inorganic-binding activitysimultaneously. The approach is a general one, and canbe used for directed immobilization of a variety of enzymesand other functional proteins and biomacromolecules with

the retained activity on desired substrates.

5. Future prospects and potentials of molecular biomimetics

The utilization of genetically engineered polypeptides asbuilding blocks and molecular tools in designing, synthesiz-ing and assembling practical systems in technology andmedicine necessitates the amalgamation of new conceptsfrom major science and technology fields (Fig. 7) [7]. Thespecific interaction between the inorganic surfaces and thepolypeptides presented by GEPIs could have a significantimpact on bio- and bionanotechnological applications,

offering several novel immediate practical advantages. The

attachment of biomolecules, in particular proteins, ontosolid supports is fundamental in the development ofadvanced biosensors [40] for the detection of a variety ofagents, and uses such as drug screening, bioseparation[41], many diagnostics applications in medicine [42] andcancer therapeutics. Protein adsorption and macromolecu-

lar interactions at solid surfaces play key roles in the perfor-mance of implants and hard-tissue engineering. Proteinsand DNA adsorbed specifically onto probe substrates areused to build micro- to nano-arrays suitable for genomics,pharmacogenetics and proteomics applications [4345].Finally, engineered polypeptides can be hybridized withfunctional synthetic molecules and nanoparticles, and thusbe used as heterofunctional building blocks in nanoscaleelectronics, magnetics and photonics. In these fields, themajor challenge to obtaining the desired effects, i.e. control-ling particle separations, organizations and distributions,can be overcome by utilizing the peptides for multifunction-ality via the genetic engineering tools.

The future use of biological materials and systems willdepend on how readily the overlap of the physical and bio-logical sciences is established [4650]. Nature has manyexamples that offer lessons for solving challenging practicalengineering problems. Not only does biology offers designlessons, but it also provides practical molecular tools tosynthesize hybrid systems [49,50]. By making useful mate-rials using genetically engineered peptides, the multidisci-plinary field of molecular biomimetics could provide anew platform for achieving the goal of efficient utilizationof biological principles by simultaneous integration of sci-ence, engineering and technology [7,17]. The coming years

will inevitably show the impact of this new field in a range

Fig. 7. Research in molecular biomimetics is multidisciplinary and provides molecular tools for applications in diverse fields of engineering and medicine.



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of diverse areas from materials to medicine, as depicted inFig. 7.

Acknowledgements

This work is sponsored by the USA-Army Research Of-

fice, through a DURINT (Defense University ResearchInitiative on NanoTechnology) Program, and by the Na-tional Science Foundation through a Materials ResearchScience and Engineering Center Program. We thank ourcolleagues S. Brown (University of Copenhagen, Den-mark), F. Baneyx, D. Schwartz, A.K.-Y. Jen and R.Samudrala for their collaborative work and invaluable dis-cussions, and our group members, E.E. Oren, T. Kacar, D.Sahin, S. Dincer, M.H. Zareie, M. Hnilova, C. So and H.Fong, for technical help.

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