Chapter 11
Protein-Mediated Bioinspired Mineralization 1,2,6
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Siddharth V. Patwardhan , Kiyotaka Shiba , Christina Raab , Nicola Huesing , and Stephen J. Clarson 5
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Department of Chemical and Materials Engineering, University of Cincinnati, Cincinnati, O H 45221 Department of Materials Science and Engineering, University of Delaware, Newark, DE 19711 Department of Protein Engineering, Cancer Institute, Japanese Foundation for Cancer Research and CREST/JST, Toshima, Tokyo 170-8455, Japan Institute of Materials Chemistry, Vienna University of Technology, Getreidemarkt 9, A-1060 Vienna, Austria Current Address: Division of Chemistry, School of Science, Nottingham Trent University, Nottingham NG11 8NS, United Kingdom *Corresponding author: Email:
[email protected]; Fax: 1-513-556-3473
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Biomineralisation, due to its remarkable sophistication and hierarchical control in mineral 'shaping', is profoundly inspiring for the design of novel materials and new processes. Recent investigations have begun to elucidate the specific interactions of various biomolecules with their respective minerals in vivo. In order to better understand the roles of such (bio)molecules in (bio)mineralisation, various in vitro model systems have been examined recently. Here we report the bioinspired mineralisation of silica in the presence of a lysine and arginine rich α-helical synthetic protein YT320 that was tailor-made using genetic engineering. Furthermore, in order to examine the possible specificity (or perhaps the lack thereof) of this protein with silica, we also report studies of bioinspired mineralisation of germania. It is proposed that this protein facilitates and 'guides' the mineralisation through residue specific and conformationally directed interactions at the molecular level.
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© 2005 American Chemical Society
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Introduction Biomineralisation* involves a complex combination of genetically controlled processes carried out in vivo that lead to the formation of highly ornate hierarchical biomineral structures that are highly species specific [1]. As an example, Figure 1 shows the fascinating beauty of patterned biosilica frustules of the diatom Aulacoseira granulata. The remarkable elegance of Nature is further illustrated by the fact that these processes typically occur under ambient conditions, unlike the corresponding laboratory methods where harsh conditions are often employed. Recent studies of the precise control of structure formation as seen in biomineralisation are beginning to reveal the possible role(s) of biomacromolecules at the molecular level [2-6]. For example, proteins have been isolated from the biogenic silica of grasses [3], sponges [4a], and diatoms [5] and were characterised. These proteins have been shown to facilitate silicification in vitro and thus it has been proposed that they are responsible for facilitating biosilicification in their respective species. These and many other
Figure 1. SEM image of the ornate nanostructured biosilica of the diatom Aulacoseira granulata. The diatom samples were collected by Professor Miriam SteinitzKannan (Northern Kentucky University) from the Ohio River (USA) on August 7, 2002. Bar = 1 μήι.
152 findings have led to the development of bioinspired in vitro model systems where the role(s) of several (bio)macromolecules in mineralisation have been investigated. Such research provides insight into biomineralisation, allows for the development of novel materials, and leads to the discovery of new material processing technologies. Indeed, the interactions of various polypeptides, polyamino acids, block copolypeptides, block copolymers and polymers with various minerals in the process of mineralisation have been documented in the cases of systems such as barium-, calcium-, lead- and silicon-based minerals [4b,7-25]. As to the role of (bio)macromolecules in bioinspired and biomimetic mineralisation, various mechanisms have been proposed [5,8,19-21,24,25]. However, the exact nature of the interactions, such as any correlation between the properties of (bio)molecules and (bio)mineral morphogenesis; and any specificity between (bio)molecules and corresponding (bio)minerals, are still unclear and thus constitute a major scientific challenge. In the case of biosilicification, proteins that are thought to direct biosilica deposition were found to contain high compositions of amino acids in the primary sequence carrying basic substituents [3a,5]. These residues are able to interact with the growing biosilica. In vitro studies with basic polyamino acids (e.g. polylysine and polyarginine) were found to affect the silicification kinetics and the structure of the resulting silica [8,9,14,19-21,23]. To date very little is known about the exact structure and behaviour of proteins isolated from living organisms that deposit biominerals and thus a clear and complete understanding of the mechanisms behind mineralisation remain to be unveiled. pYT320 MRGSHHHHHHSSGWVD PENLQAE RKVLQGRMENLQAE RKVLQGRMENLQAE RKVLQGRMENLQAE RKVLQGRMENLQAE RKVLQGRMENLQAEP QSIAGSYGKPASGG
MW 12.3 pi 9.86 alpha-rich in CD
Figure 2. Amino acid primary sequence of the YT320 protein.
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In this research, we have carefully and intentionally chosen to investigate the use of a lysine and arginine rich protein YT320 of known and well defined secondary structure (Figure 2). In particular, we report the bioinspired mineralisation of silica in the presence the protein YT320. As it is known that germanium can be incorporated into biogenic silica; that the chemical characteristics of germanium and its resemblance to silicon have been described previously; and that it competes with silicon in biosilicification[26], it is our hypothesis that the various (bio)macromolecules that facilitate (bio)silicification may also influence related mineral formation in vitro, as shown in Scheme 1. Furthermore, it should be noted that although naturally occurring proteins [27], model peptides (e.g. the R5 peptide derived from the diatom Cylindrotheca fusiformis [5,7,15]), polyamino acids [8,9,14], and block copolypeptides [4b] have been studied in their respective in vitro model systems, the in vitro mineralisation utilising genetically engineered synthetic proteins has not been studied to date.
Experimental The protein YT320 was prepared by tandem polymerisation of the 42-bp long microgene, MG-15, whose first coding frame had similarity to a segment of the coiled coil α-helix of the natural protein (seryl-tRNA synthetase). The
Scheme 1. Silica formation and structure control has been observed in vivo (right top) and in vitro (right center); and the role(s) of (bio)macromolecules is being studied. Due to similarities in the sol-gel chemistry of silica and germanium dioxide, can the same (bio)macromolecules be used for structure formation ofGe0 ? 2
154 protein possessed a calculated pi of 9.86 and thus it is cationically charged at circumneutral pH (i.e. under the reaction conditions employed here). The protein YT320 has 5.5 repeats of the α-helix forming frame of the microgene, comprising about 80% of the protein and showed a typical α-helix-rich spectrum in Circular Dichroism (CD) analysis [28a]. Small Angle X-ray Scattering analysis has shown that protein YT320 does not fold as tightly as natural proteins but is rather more compact than if completely denatured. The protein has been crystallised, indicating repetitious artificial proteins can undergo a transition to a more ordered state by choosing appropriate conditions [28b]. Tetramethoxysilane (TMOS), used as a silica precursor, was obtained from Sigma-Aldrich. Water glass or sodium silicate (>27% as Si0 ) was purchased from Riedel-de Haën. Ethylene glycol modified silane (EGMS) was synthesised as described below. For the preparation of EGMS, tetraethoxysilane (TEOS) was mixed with a four-fold molar excess of ethylene glycol and heated to 150° C under argon. Continuous distillative removal of ethanol resulted in the formation of a clear oily liquid product [29]. This novel water soluble precursor does not require any catalyst for hydrolysis and condensation. In addition, ethylene glycol released upon hydrolysis of EGMS does not appear to be detrimental for biomolecules [30]. Precursors used for germania synthesis were germanium(IV) ethoxide and germanium(IV) isopropoxide and they were obtained from SigmaAldrich. Potassium phosphate buffer (pH 7) was used to maintain the reaction mixture at neutral pH and was obtained from Fisher. Bioinspired silica formation assay was performed in the presence of the YT320 protein, starting with several silica precursors as listed in Table 1. Upon mixing a precursor solution with a protein solution of the desired concentration and buffering the reaction content to pH 7, silica precipitation was observed. The synthetic procedures were similar to those we have previously described [13,19,20]. Briefly, in potassium phosphate buffer (pH 7), 5 mg/ml of the YT320 protein solution (in the same buffer) was added and then 1 M pre-hydrolysed TMOS solution was added. The volume ratios of buffer: protein solution: silica precursor solution was 8:2:1 in the case of TMOS based synthesis. For the experiments under externally applied shear, the TMOS based reaction mixture was stirred for 5 minutes. The product was then centrifuged, washed and dried. 2
Table 1. Silica precursors used for the silicification reactions. Silica Precursors Tetramethoxysilane (TMOS) Water glass (sodium silicate) Ethylene glycol modified silane (EGMS) fl
a
Structure Si(OCH )4 Na Si 07 3
2
\
O
3
P^y^oH Si' OH
0
TMOS and water glass were pre-hydrolysed.
155 When EGMS was used as the silica precursor, 3 pL EGMS was directly added to a mixture of 20 of a 5 mg/ml YT320 protein solution and 80 μL· buffer. In the case of water glass as the silica precursor, 1 ml water glass was hydrolysed in 1 mM HC1 before use. 10 pL of this water glass solution was mixed with 20 pL protein solution (5 mg/ml) and was then buffered. After 5 minutes of reaction time in each case, the solution was centrifuged and washed with DI water three times. A few drops of washed solutions were placed on sample holders in each case and left to dry under ambient conditions. The dried samples were either coated by evaporation of gold-palladium alloy or sputter coated with gold and they were further characterised by Field EmissionScanning Electron Microscope (FE-SEM) for product morphology, which was also equipped with an Energy Dispersive Spectroscopy (EDS) facility for elemental analysis. In the control experiments without using the protein, no silica precipitation was observed even over a 24 hour period, as was described previously [13]. For germania synthesis, 2 μί, of either germanium (IV) ethoxide or germanium (IV) i-propoxide was directly added to a mixture of 20 pL of a 5 mg/ml YT320 protein solution and 80 pL buffer. The mixture was left for 5 minutes to react and was then washed and characterised as described above. No precipitation was observed in control experiments carried out without any protein.
Figure 3. Representative SEM images of YT320 mediated silica particles synthesised using pre hydrolysed TMOS under static conditions. Bar = 1 μπι.
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Figure 4. Elongated silica structures using pre-hydrolysed TMOS and YT320 formed upon subjecting the medium to shear for five minutes. Highlighted areas from (a) and (b) are presented at higher magnifications in (b) and (c) respectively. Bar = 1 pm.
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Results and Discussion To our knowledge this is the first attempt demonstrating the use of a genetically engineered synthetic protein for mineralisation of silica and germania. The results obtained for each respective system are presented and discussed below. The SEM analysis of the silica particles revealed that spherical or sphere like particles were successfully synthesised (Figure 3). In the case of the tetramethoxysilane (TMOS) based system, well-defined spherical silica particles were observed with average sizes of approximately 300 nm. Upon subjecting the reaction mixture to the externally applied shear, ellipsoidal (Figure 4), fibre-like (Figure 5a, b) and fused (see arrowhead in Figure 5c) particles were seen in addition to spherical particles. The formation of non-spherical elongated morphologies as observed herein is in accordance with our previous investigations for related systems [7,22]. The (bio)macromolecule conformation in solution may be responsible for the formation of such elongated structures [10,11]. When ethylene glycol modified silane (EGMS) was used as the silica precursor, similar to the TMOS based system, formation of approximately 300 nm sized spherical silica particles were synthesised (see Figure 6). Formation of
Figure 5. Fiber-like silica structures using pre-hydrolysed TMOS and YT320 formed upon subjecting the medium to shear for five minutes. Highlighted area in (a) is presented at higher magnification in (b). The arrowhead in (c) indicates the formation of 'fused' structures. Bar = / pm.
158 interconnected networks were observed instead when water glass was used as the silica precursor. The formation of silica was further confirmed by silicon and oxygen signatures as observed in Energy Dispersive Spectroscopy (EDS) (Figure 6). The incorporation of YT320 into silica was also verified by the carbon and nitrogen peaks in the EDS. The aluminium and magnesium peaks correspond to the sample holder while the phosphorous peak may be due to the buffer used. In order to extend our investigations on the role(s) of (bio)macromolecules in bioinspired mineralisation, we have studied the in vitro mineralisation of germania in presence of several (bio)macromolecules. The YT320 protein (reported herein) was found to facilitate germania particle formation (Figure 7). It is noted that no precipitation was observed in the absence of the protein. The elemental analysis confirmed the presence of germanium and oxygen indicating the formation of germania. The carbon and nitrogen signatures suggest the occlusion of the protein. In addition, a synthetic polymer (polyallylamine hydrochloride), that has been previously studied for silica precipitation, was also found to direct the formation of germania in vitro, thus further supporting our hypothesis [31]. In a recent study, Morse and co-workers have shown that the silicatein protein isolated from the silica spicules of sponge Tethya aurantia was able to catalyse the formation of titania [32]. It was further reported that silicatein may stabilise anatase - a polymorph of titania that is typically formed only at higher temperatures in the absence of silicatein protein. Their findings on silicatein mediated titania synthesis and the data presented in this paper on the
Figure 6. (a), (b) SEM and (c) EDS images of YT320 mediated silicification using EGMS as the silica precursor. The highlighted area in (a) is presented at higher magnification in (b). Bar = 500 nm.
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Figure 7. Representative SEM micrograph and EDS spectrum for germania prepared by YT320. Bar = lpm. role of a genetically engineered synthetic protein in the formation of silica and germania demonstrates the ability of such biomolecules to be tailored for mineralisation and thus proven to be technologically important, as hypothesized previously [20]. Metal alkoxides undergo polymerisation via hydrolysis and condensation to produce oligomers. These oligomers then grow in size and they act as nuclei. Particle formation occurs due to the growth in size of these nuclei either by precipitation of soluble species (monomers and dimers) or by coagulation of insoluble particles. At circumneutral pH, the particles possess a net negative surface charge. As mentioned above, the protein YT320 possessed a pi of 9.86
160 and thus it is cationically charged at pH 7 - the reaction conditions employed here. It is thus proposed that the protein under consideration here interacts electrostatically with the minerals at molecular level. It is interesting to note that only some biomolecules have an effect on mineralisation, while many others lack such ability as demonstrated by Livage and co-workers recently [33]. Hence the role to YT320 in successfully facilitating mineralisation as presented herein is of particular importance. The activity of YT320 protein in the in vitro bioinspired mineralisation is proposed to be due to the organisation of the protein molecules in solution. This spatial arrangement is then followed by nucleation, catalysis and/or scaffolding of the inorganic structures around the protein molecules leading to phase separation as described in detail elsewhere [5,8,19-21,24-25]. This model further predicts that the three-dimensional organisation of such (bio)molecules in solution may govern the structures of the respective (bio)minerals which may be in turn controlled by chemical structure of the (bio)molecules. In vivo, as the primary sequence of proteins (i.e. chemical structure) and their structure and functions are genetically controlled, the formation of species-specific biomineral structures is presumably genetically governed. It should be kept in mind at this point that this simple model for the interactions of (bio)molecules and (bio)minerals is only a first approximation. Various parameters affecting biomineralisation in vivo such as the presence of other (macro)molecules, pH and concentration (chemical potential) gradients have yet to be fully considered. The results discussed herein on silica and germania synthesis in the presence of a carefully tailored protein supports our earlier hypothesis [20] that the effect of (bio)macromolecules on in vitro mineralisation can be somewhat generic rather than being specifically limited to a particular mineral system. Various proteins of known sequence and structure are currently being investigated for their role in mineralisation. In addition, further studies pertaining to the solution behaviour of such (bio)molecules, their interactions with inorganic species at the molecular level and their exact roles (catalytic or scaffolding or both) in mineralisation are currently being undertaken [34].
Conclusions We report here for thefirsttime, an investigation of a genetically engineered protein in mediating the bioinspired mineralisation of silicon-based and germanium-based systems. Ionic and hydrogen-bonding interactions between the organic and inorganic molecules in addition to chain conformations and selfassembly of the protein are thought to be key in such bioinspired mineralisation.
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Acknowledgements We would like to thank Professor Miriam Steinitz-Kannan for kindly providing us with the diatom sample. S. V. P. thanks Professor Kristi L. Kiick and Professor Carole C. Perry for various helpful discussions. K. S. thanks Ms. Tamiko Minamizawa for her technical assistance.
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