Molecular Biomimetics: Linking Polypeptides to Inorganic Structures

In developing novel materials, Mother Nature gave us enormous inspiration with its already existing highly organized structures varying from macro to nanoand molecular scales. Biological hard tissues are the examples of composite hybrid materials having both inorganic and organic phases that exhibit excellent physical properties, all based on their evolved architectural design. Biocomposites incorporate both structural macromolecules, such as proteins, lipids and polysaccharides and minerals, such as hydroxyapatite, silica, magnetite, and calcite. Among these, proteins are the most instrumental components for use in materials fabrication because of their molecular recognition, binding and self-assembly characteristics. Consequently, based on this premise, inorganic surface specific polypeptides could be a key in the molecular engineering of biomimetic materials. Peptides can now be selected by directed evolution, adapted from molecular biology, by using combinatorial peptide libraries, analogous to natural selection. Adapting genetic approaches further allow to redesign, modify or engineer the selected first generation peptides for their ultimate utilization in bionanotechnological applications as molecular erectors, couplers, growth modifiers and bracers. Introduction Mother Nature has provided a high degree of sophistication in materials and systems at the nanometer scale. Naturally occurring materials have remarkable functional properties derived from their highly organized structures from the molecular to the nano-, micro-, and macroscales, with intricate architectures (Fig. 8.1). They are self-directed in their organization and formation, operate in water environment, dynamic in their interaction with the surroundings, complex in their structures and functions self-healing in damage control. Yet, they are not achievable in purely synthetic systems under the same efficient energy conserving, no waste delivering manner (Lowenstam, 1989; Sarikaya, 1999; Ball, 2001; Sanchez et al., 2005). With the integration of recent developments in molecular and nanoscale engineering in physical sciences, and the advances in molecular biology, materials fabrication through biology, biomimetics, is now entering the molecular scale (Sarikaya et al., 1995; 2003). Utilizing closely controlled molecular, nanoand microstructures through molecular recognition, templating and self assembling properties of Nature, molecular biomimetics is evolving from the true marriage of physical and biological sciences (Niemeyer, 2001; Sarikaya et al., 2004). 1 | Tamerler and Sarikaya Biological hard tissues are the examples of composite hybrid materials having both inorganic and organic phases and exhibiting excellent physical properties thereby creating ecological intakes for the host organisms (Mann 1996; Mann et al., 1998; Ball, 2001). Biocomposites have incorporated both structural macromolecules such as proteins, lipids and polysaccharides and minerals, such as hydroxyapatite, silica, magnetite, and calcite (Berman et al., 1988; Ratner et al., 1996; Cha et al., 1999; Mayer et al., 2002). Among these, proteins are the most promising molecules because of their recognition, binding and self assembly characteristics. The advantage of a molecular biomimetic approach to nanotechnology, therefore, is that inorganic surface-specific proteins could be used as couplers, growth initiators and modifiers, brazers and molecular erector sets, for self assembly of materials with controlled organization and desired functions. The realization of heterofunctional nanostructure materials and systems could be at three levels, all occurring simultaneously feed backing each other as the Mother Nature produces her materials and components. The first is that the inorganic specific peptides are identified and peptide/protein templates are designed at the molecular level through directed evolution using the tools of molecular biology. This ensures the molecular-scale up processing for nanostructural control at the lowest dimensional scale possible. The second is that these peptide building blocks can be further engineered to tailor their recognition and assembly properties similar to the Nature’s way of successive cycles of Figure .1 Examples of biologically fabricated complex nanomaterials. (A) Layered nanocomposite: growth edge of nacre (pearl) of abalone (Haliotis rufescens): Aragonite platelets separated by a thin-film of organic matrix. (B) Nanomagnetics: magnetite (Fe3O4) particles in magnetotactic bacteria: Aquaspirillum magnetotacticum. (C) Hierarchical structure: 3D woven enamel rods of hydroxyapatite crystallites of mouse teeth. (D) Biofiber-optics: a layered siliceous spicular optical fiber of a sponge (Rosella) and its apex (inset), novel design of a lens, a light collector. | 1 Tools for Bionanotechnology mutation and generation can lead to progeny with improved features eventually for their utilization as couplers or molecular erector sets to join synthetic entities, including nanoparticles, functional polymers, or other nanostructures onto molecular templates (molecular and nanoscale recognition). Finally, the third is that the biological molecules selfand coassemble into ordered nanostructures. This ensures an energy efficient robust assembly process for achieving complex nano-, and possibly hierarchical-structures, similar to those found in Nature (self-assembly) (Sarikaya et al., 2004). There are different ways to obtain the inorganic surface specific proteins such as extraction from hard tissue, designing them via theoretical approaches or utilizing the limited number of already existing ones (Carlolou et al., 1988; Paine et al., 1996; Schneider et al., 1998; Kroger et al., 1999; Cha et al., 1999; Liou et al., 2000). Each of these approaches has its own major limitations and may not be practical enough to serve in all nanoscale-engineering applications. Inorganic surface specific peptides could be the key in the molecular engineering of bioinspired materials. However, there are only a few polypeptides have been identified that specifically bind to the inorganics. With the recent developments in recombinant DNA technology, these inorganic surface specific proteins can now be designed, modified or engineered for the production of nanostructured materials. During the last decades, combinatorial biology based molecular library systems have been developed for selecting substratespecific peptide units, mostly for medical applications but only recently they are applied for selecting short peptides for inorganic surfaces (Brown, 1997; Whaley et al., 2000; Gaskin et al., 2000; Naik et al., 2002; Sarikaya et al., 2004). In these library systems, polypeptides are the major displayed molecules, which can be screened for the specific properties. In the following sections, we provide an overview of molecular biomimetics approaches to achieve the premises of nanotechnology and summarize its potentials and limitations. Then, we look into the ways finding polypeptides that recognize inorganics, and describe the protocols of combinatorial biology for identifying, characterizing and engineering peptides to utilize them as molecular buildings blocks of future bimimetic materials and systems. Here we emphasize on the cell surface and phage display technologies that are well adapted for the identification of inorganic surface specific peptides, and to further tailor the characterized peptides using postselection engineering. We then discuss the possible mechanisms through which a given protein might selectively bind to an inorganic based on their thoroughly binding characterization. We present examples of current achievements in utilizing engineered polypeptides are given to demonstrate their potential use and, finally, we present future prospects of molecular biomimetics in bioto nanotechnologies. Potentials and limitations of nanotechnology The fundamental premise in the field of nanotechnology has been that the length scales, which characterize materials structure and organization, predominantly determine their physical properties (Drexler, 1992; Schmid, 1994; Ferry et al., 1997; Katz et al., 2004). Mechanical properties of nanocomposites, light harvesting properties of nanocrystals, stain defender properties of nanoparticles, magnetic properties of single-domained particles, barrier properties of nanoclays to extend the shelf lifes of bottles, and solution properties of col1 | Tamerler and Sarikaya loidal suspensions are all examples to show that nanotechnology is not a futuristic technology, it is already establishing its place in our daily life ( Jackson et al., 2002; Shipway et al., 2001; Hoenlein et al., 2003; Thayer et al., 2004). All of the given examples correlate directly to the nanometer-scale structures that characterize these systems. In building the nanometer scale structures, the approach is to design molecule by molecule with a purpose such as developing an advanced nanoscale machine or assembler or fabricator. Once, the materials are at the nanoscale then they present unique characteristics based on physical phenomena, and therefore, the physical and chemical rules governing the macroscale materials might not reflect their displayed properties (Ferry et al., 1997; Muller, 2001). Recent experimental research in the field of nanometer-scale electronics and photonics has confirmed theoretical predictions in molecular and nanometer-scale structures, e.g. organized quantum dots, and electrical transport in nanotubes and wires. In addition, colloidal particles of metals, functional ceramics, and semiconductors have potentially useful electronic, optoelectronic and magnetic properties that derive from their small size. These properties may lead to their application as chemical, biological, and optical sensors, spectroscopic enhancers, nanoelectronics, and quantum structures, among others (Harris, 1999; Gittins et al., 2000; Bachtold et al., 2001; Huang et al., 2001; McDonald et al., 2005). Successful integration of nanoscale materials to technology requires creation of millions of these structures in parallel (Glotzer, 2004). The conditions for controlled structures at nanometer scale can be obtained by promoting the self-assembly nature of the molecules through balancing kinetic and thermodynamic forces, yet this does not provide a specific geometry or functionality. One way to overcome this problem is to combine “self-assembly” with more conventional “bottom-up technology” to provide suitable functionalities with specific structures. However, there a number of challenges to be overcome such as if the structures available from “self-assembly” technique can provide functionality comparable to that realized by “bottom-up” process, or if the architectures can be built by the choice of material functionality rather than the availability of materials which are applicable to the system (Seeman et al., 2002; Sarikaya et al., 2004) Self-assembled layers are often demonstrated using thiol-derived molecules on gold or silanes on oxides. This is because the sulfur or hydroxyl atoms chemisorb to the gold or silica surface, respectively. This is advantageous for the gold-or silica-based architectures. However, more practical approach to multifunctional materials is to use substrates other than gold or silica. Availability of new materials will extend current technology with biosorption in addition to traditional chemisorption. The realization of the full potential of nanotechnological systems has so far been limited because of the difficulties in their controlled-synthesis and the subsequent assembly into useful functional structures and devices. Most traditional approaches to synthesis of nanoscale materials are energy inefficient, require stringent synthesis conditions, and often produce toxic byproducts. These techniques still use “top-down” approaches, and even the most advanced microtechnology and recently developed nanotechnology, such as self-assembly through chemistry, in-jet technology, dip-pen lithography, and microcontact printing, require considerable external manipulation that curtail the achievement of complex 3D architectures and robust scale-up, and, hence, limit the potential | 1 Tools for Bionanotechnology of nanoscale related physical properties (Gooding et al., 2003; Quist et al., 2005). Furthermore, the quantities produced are small and the resultant material is often highly irreproducible because of uncontrolled agglomeration. Even in the case of carbon nanotubes, one of the most successful nanotechnological materials, there are still some practical limitations to their widespread use, including uniformity, and control of surface chemistry, and for twoand three-dimensional assembly (Harris, 1999; Hoenlein et al., 2003). Despite all the promise of science and technology at the nanoscale, the control of nanostructures and ordered assemblies of materials in twoand three-dimensions remains not fully accomplished. Inspiration from Nature for realization of nanotechnology Mother Nature has been an inspiration in fully achieving the promises of nanotechnology. The key to nanotechnology is primarily to understand how nature works at the highest level of sophistication with efficient energy use without waste accumulating way (Sarikaya, 1999; Ball, 2000; Seeman et al., 2002). Biomaterials are highly organized from the molecular to the nano-, micro-, and the macroscales, often in a hierarchical manner with intricate nanoarchitectures that ultimately make up a myriad of different functional units, soft and hard tissues. Hard tissues such as bones, dental tissues, spicules, shells, bacterial nanoparticles can be given as examples which all have one or more protein based components ( Carilolou et al., 1988; Berman et al., 1988; Schultze et al., 1992; Kaplan et al., 1994; Paine et al., 1996; Fallini et al., 1996). The inorganic part could be the magnetite (Fe3O4) nanoparticules in the case of magnetotactic bacteria to sense the direction of gravity with many different morphologies depending on the type of species, or it could be silica forming the sponge spicule to serve as light collector, or hydroxyapatite crystals in enamel providing 3D woven enamel rod structure or the aragonite platelets in abalone shell to present the microarchitecture (Fig. 8.1) (Fong et al., 2000; Sarikaya et al., 2001; 2003; 2004). They are simultaneously selforganized, dynamic, complex, self-healing, and multifunctional, and have characteristics difficult to achieve in purely synthetic systems even with the recently developed bottom up processes. Based on their closely controlled nanostructures achieved through molecular recognition, templating, and self-assembly, biological materials have properties of technological interest that surpass synthetic systems with similar phase compositions. Under genetic control of the organisms, biological tissues are synthesized in aqueous environments in mild physiological conditions using biomacromolecules, primarily proteins but also carbohydrates and lipids. Proteins both collect and transport raw materials, and consistently and uniformly selfand coassemble subunits into shortand long-range ordered nuclei and substrates. Whether in controlling tissue formation, participation in its formation, or being integral part of the tissue in its biological functions and physical performance, proteins are indispensable part of the biological structures and systems. A simple conclusion is that any future biomimetic system, whether for biotechnology or nanotechnology, should include protein(s) in its assembly and, perhaps, in its final structure. Engineering materials, containing one of more phases, are synthesized via a combination of approaches using, for example, melting and solidification processes that are often followed by thermomechanical 1 | Tamerler and Sarikaya treatments, or solution/vacuum deposition and growth processes and finally annealing. Chemical recognition and synthetic selfassembly processes are a step beyond these traditional approaches. Many examples have been shown in the last decade showing these processes can produce highly ordered and predictable structures, including, for example, mesoporous systems based on surfactant/ceramic precursor molecules; self-assembled monolayers, and hybrid macromolecules. In many cases, however, the final product is a result of a balance of interactions, dictated by the kinetics and thermodynamics of the system, that are often achieved through “heat-and-beat” approaches of traditional materials science and engineering (Sarikaya et al., 1982; DeGarmo et al., 1988). In biological systems, the same balance is achieved through evolutionary selection processes that result in the emergence of a specific molecular recognition. For example, in antigen/antibody interactions, lock-and-key is one of the main mechanisms by which two molecules specifically recognize each other. In the new field of molecular biomimetics, hybrid materials could be assembled from the molecular level using the recognition properties of the proteins that specifically bind to the inorganics. Using the peptide based molecular approaches, new generation of binding agents, couplers, or molecular erector sets could be designed for self assembly of materials with controlled organization and specific functions. The current state of protein folding prediction and surface binding chemistry do not provide sufficiently detailed information to perform rational design of these hierarchical structures. To circumvent this problem, massive libraries of randomly generated peptides can be screened for binding activity to inorganic surfaces via the use of phage and cell surface display techniques. In either case, it may ultimately be possible to construct a “molecular erector set” in which different types of proteins, each designed to bind to a specific inorganic surface, could assemble into intricate, hybrid structures composed of inorganics and proteins. Below we demonstrate the general approaches with some examples. Combinatorial biology approach in selecting inorganic-specific peptides Combinatorial strategies in chemistry and biology have attracted great interest to search and generate active molecular compounds for various applications over the last decades. The discovery of the counter parting active molecule from a series or a large number of mixtures has been revolutionizing idea brought by the combinatorial methods. First, combinatorial chemistry-based methods started with large peptide libraries following the establishment of solid phase synthesis of peptides. Optimization and rapid development of parallel syntheses and automation resulted in screening large number of compounds for a particular pharmaceutically interesting property such as increasing selectivity, activity or lowering toxicity (Beck-Sickinger et al., 2002). Later, the integration of combinatorial methods into biological selection strategies have brought advantages over the chemistry based ones since they utilize the production capacities of the living systems, phages and cells. Over the last 10 years, various methods using organisms have been established to produce large libraries of peptides, proteins or nucleic acids. These libraries were generated both in vivo environments where organisms such as cells and viruses have been utilized, and in vitro environments where biological molecules key to synthesis, have | 1 Tools for Bionanotechnology been added into the reaction mixtures. The generated molecules are directed towards a certain target interaction where they are enriched and identified according to their desired property. The idea of searching for its own active molecule is not new considering biological interactions, such as receptors on cell surfaces recognize their ligands among many different molecules or antibodies detect the certain fragments of bacterial or viral surface proteins. These high-throughput strategies are only bringing us closer to Mother Nature’s ways in developing new molecular tools. Nature’s building blocks indeed are based on simple elements; ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) are formed through the polymerization of four different nucleotides and peptides or proteins are formed through the condensation reaction of 20 different natural amino acids. For a nucleic acid that is 300 base pair in length or composed of 100 codons, there would be a possibility of translating into 20100 different proteins. In combinatorial biology based library systems, polypeptides have been the major biological compounds because of their very efficient recognition properties at the nanometer scales. In this section, we will go over the basic principles of display technologies and discuss phage and cell surface display methods in detail, starting from their use as a tool for protein–protein interactions, then their adaptation for protein–inorganics interaction over viewing both advantages and drawbacks of the each display system. Principle of display technologies Display technologies, in general as a routine tool, refers to a collection of methods for creating libraries of biomolecules that can be screened for desired or novel properties, by optimizing the assembly of building blocks with more diverse function. Since the invention of phage display nearly two decades ago, display technologies have proven to be an extraordinarily powerful tool for a various biotechnological and biological applications (Smith, 1985; Dani, 2001; Benhar, 2001; Ma et al., 2001; Mrskich, 2002; Wernerus, 2004). Mainly protein–protein interactions were studied in a variety of contexts including characterization of receptor and antibody binding sites, ligand specificities, and the isolation and evolution of proteins or enzymes exhibiting improved or otherwise altered binding characteristics for their ligands. Biological libraries composed of peptides, antibodies, or proteins can be displayed by using either in vivo or in vitro display technologies. Regardless of the display technologies, there are three major components in the system: displayed molecule, its genetic code and a common linker to the displayed system. Following the interaction of the library with the counterpart molecule, high-throughput selection of the desired molecules with the possible specificity and affinity towards the counterpart molecule is carried out. All technologies are based on the common theme of cloned gene and its encoded protein is physically linked; therefore, the genetic information translating to the protein with the desired phenotypic character could be accessed easily. In in vivo display, stable genes are expressed either through transfection or introduction of foreign DNA into the cells, whereas in vitro systems cell free extracts will transcribe the cloned template (Hoess, 2001, Samuelson, 2002). Consequently, in vitro systems are not limited by the transformation efficiency of a cellular host (Dower et al., 2002; Lipovsek et al., 2004). In in vivo display technologies, biological host can be phage, such as very well established filamentous bacteriphage 1 | Tamerler and Sarikaya M13, or alternative ones as λ, T4 or T7 phage, or cells, including prokaryotes and eukaryotes. In phage display, the coat protein genes are used to display the molecular library, whereas cell wall or periplasmic display systems are successfully applied to expression of molecular libraries. Outer membrane proteins, lipoproteins, fimbria, and flagellar proteins can be used for heterologous surface display on bacteria. With the data from human genome project, now determining the functions of the proteins is an important task especially for diagnosis and therapeutics. Mammalian cell surface expressions of receptor or transmembrane proteins have been attempted for being displayed on the current systems, however low efficiency of cloned gene delivery is a major problem. Many viral expression-cloning systems have been still continually searched for displaying mammalian proteins interactions in their in vivo environment. Among eukaryotic cells, yeast two hybrid systems have been very promising for expressing eukaryotic proteins (Ueda, 2004). However all of these systems still carry the limitations living cells such as rather restricted library size or suppression of certain mutant by the molecular machinery of the host or even sometimes they are not correctly folded or transported not contributing to the library diversity. Ribosome, mRNA and DNA display technologies are developed as cell free protein synthesis systems to overcome the limitations brought by the transformation efficiency of a cellular host, therefore they can be operated in the absence of a living cell (Fitzgerald, 2000; Amstatz et al., 2001). Consequently, they can present high library size and also unique applicability to directed evolution of proteins, since selection with these systems can also be performed under the biologically incompatible environments (Takahashi, et al., 2003).