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Nanosilica Formation At Lipid Membranes Induced by the Parent Sequence of a Silaffin Peptide Michael S. Kent,† Jaclyn K. Murton,† Frank J. Zendejas,† Huu Tran,† Blake A. Simmons,*,† Sushil Satija,‡ and Ivan Kuzmenko§ Sandia National Laboratories, Albuquerque, New Mexico, Sandia National Laboratories, LiVermore, California, National Institute of Standards and Technology, Gaithersburg, Maryland, and AdVanced Photon Source, Argonne National Laboratories, Argonne, Illinois ReceiVed March 20, 2008. ReVised Manuscript ReceiVed September 17, 2008 Diatoms are unicellular eukaryotic algae found in fresh and marine water. Each cell is surrounded by an outer shell called a frustule that is composed of highly structured amorphous silica. Diatoms are able to transform silicic acid into these sturdy intricate structures at ambient temperatures and pressures, whereas the chemical synthesis of silicabased materials typically requires extremes of temperature and pH. Cationic polypeptides, termed silica affinity proteins (or silaffins), recently identified from dissolved frustules of specific species of diatoms, are clearly involved and have been shown to initiate the formation of silica in solution. The relationship between the local environment of catalytic sites on these peptides, which can be influenced by the amino acid sequence and the extent of aggregation, and the structure of the silica is not understood. Moreover, the activity of these peptides in promoting silicification at lipid membranes has not yet been clarified. In this work, we developed a model system to address some of these questions. We studied peptide adsorption to Langmuir monolayers and subsequent silicification using X-ray reflectivity and grazing incidence X-ray diffraction. The results demonstrate the lipid affinity of the parent sequence of a silaffin peptide and show that the membrane-bound peptide promotes the formation of an interfacial nanoscale layer of amorphous silica at the lipid-water interface.
Introduction Diatoms are one of the most important biological systems on the planet, accounting for up to 25% of global carbon fixation.1,2 These eukaryotic organisms typically possess a highly silicified structure known as the frustule.3-7 Evidence indicates that diatoms utilize several organic components, including silica affinity peptides or silaffins, to promote silica growth during cell division.8-12 Kroger et al. isolated silaffins from the purified frustules of several species that were so tightly bound to the silica that they could only be solubilized after dissolution of the frustule with HF.8,10-12 These proteins have been shown to accelerate silica polymerization and are only expressed during cell division. The peptide termed R5 (H2N-SSKKSGSYSGSKGSKRRIL-CO2H) is the parent sequence of a much larger protein referred to as silaffin 1A (isolated from Cylindrotheca fusiformis) that is highly post-translationally modified,10 wherein * Corresponding author. † Sandia National Laboratories. ‡ National Institute of Standards and Technology. § Argonne National Laboratories. (1) Johnston, A. M.; Raven, J. A.; Beardall, J.; Leegood, R. C. Nature 2001, 412, 40–41. (2) Granum, E.; Raven, J. A.; Leegood, R. C. Can. J. Bot. 2005, 83, 898–908. (3) Hazelaar, S.; van der Strate, H. J.; Gieskes, W. W. C.; Vrieling, E. G. J. Phycol. 2005, 41, 354–358. (4) Noll, F.; Sumper, M.; Hampp, N. Nano Lett. 2002, 2, 91–95. (5) Crawford, S. A.; Higgins, M. J.; Mulvaney, P.; Wetherbee, R. J. J. Phycol. 2001, 37, 543–554. (6) Mann, S.; Ozin, G. A. Nature 1996, 382, 313–318. (7) Pickett-Heaps, J.; Schmid, A. M. M.; Edgasr, L. A. In Progress in Phycological Research; Round, F. E., Chapman, D. J., Eds.; Biopress: Bristol, U.K., 1990; Vol. 7, pp 1-169. (8) Poulsen, N.; Kroger, N. J. Biol. Chem. 2004, 279, 42993–9. (9) Kroger, N.; Deutzmann, R.; Bergsdorf, C.; Sumper, M. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 14133–8. (10) Kroger, N.; Deutzmann, R.; Sumper, M. Science 1999, 286, 1129–32. (11) Kroger, N.; Deutzmann, R.; Sumper, M. J. Biol. Chem. 2001, 276, 26066– 70. (12) Kroger, N.; Lorenz, S.; Brunner, E.; Sumper, M. Science 2002, 298, 584–6.
many of the lysines are modified to polyamines and the serines are phosphorylated. The modified lysines are important in the silica-precipitating characteristics of the protein.11,13 In addition to silaffins, long chain polyamines have also been found in the cell walls of some species.9 Both silaffins and polyamines induce precipitation of silica spheres when brought into contact with silicic acid at near-neutral pH, and there is some evidence to suggest that interactions between the two play an important role in controlling the morphology of the silica formed.8-12 The local silica-precipitating environment is influenced by the amino acid sequence and the nature of peptide aggregation, among other factors.14 Interactions between the peptides and lipid membranes may also affect the local environment of the silica-initiating sites. Several studies of protein- or polymerinduced silica formation have focused on the precipitation of silica nanospheres from solution with no lipid-based structures present.9-12,15-17 Other studies have shown that silica formation can be induced at certain lipid membranes in a controlled fashion, including silica nanoparticles generated by silica formation on the outer surface of catanionic lipid vesicles.18,19 The present work reports the adsorption of a peptide similar to R5 to lipid monolayers and the subsequent peptide-induced formation of nanosilica layers upon introduction of silicic acid. The work demonstrates that fine details of the silicification process can be revealed using planar lipid membranes combined with analysis by grazing incidence scattering methods. The results suggest (13) Sumper, M.; Kroeger, N. J. Mater. Chem. 2004, 14, 2059–2065. (14) Knecht, M. R.; Wright, D. W. Chem. Commun. 2003, 3038–3039. (15) Knecht, M. R.; Sewell, S. L.; Wright, D. W. Langmuir 2005, 21, 2058– 2061. (16) Knecht, M. R.; Wright, D. W. Langmuir 2004, 20, 4728–4732. (17) Naik, R. R.; Whitlock, P. W.; Rodriguez, F.; Brott, L. L.; Glawe, D. D.; Clarson, S. J.; Stone, M. O. Chem. Commun. 2003, 238–239. (18) Hentze, H. P.; Raghavan, S. R.; McKelvey, C. A.; Kaler, E. W. Langmuir 2003, 19, 1069–1074. (19) Lootens, D.; Vautrin, C.; Van Damme, H.; Zemb, T. J. Mater. Chem. 2003, 13, 2072–2074.
10.1021/la801794e CCC: $40.75 2009 American Chemical Society Published on Web 11/26/2008
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that peptide-lipid interactions and lipid phase behavior could be used to control silica morphology in synthetic systems. This Article concerns a peptide with the sequence YYSGSYSGSKGSKKRRILKRRIL-COOH. This sequence was chosen to contrast with R5 in two significant aspects: (1) it has two RRIL groups on the C-terminus instead of one, and (2) it has YY instead of SSKK on the N-terminus. The former was selected to better understand the effects of the RRIL sequence, which has been shown to be crucial to silica polymerization,14 and the latter was made to increase the adsorption to lipid membranes. A future report will compare the results for this peptide with results for R5 and a third peptide that lacks RRIL. The nanosilica structure was characterized in situ by X-ray reflectivity (XR) and grazing incidence X-ray diffraction (GIXD). XR provides the electron density profile normal to the interface,20 whereas GIXD is sensitive to highly ordered structures in the plane of the membrane.21 The results show that this peptide associates strongly with, and inserts into, membranes of 1,2-dipalmitoyl-sn-glycero3-[phospho-rac-(1-glycerol)] (DPPG) and induces formation of a silica layer in the presence of silicic acid.
Experimental Section Materials. DPPG was purchased from Avanti. The peptide with sequence YYSGSYSGSKGSKKRRILKRRIL-COOH was purchased from SynBioSci Corporation (Livermore, CA). Molecular weight characterization by mass spectrometry was provided by the manufacturer and indicated 95% purity. Chloroform (ethanol stabilized) and sodium metasilicate were purchased from Aldrich. All materials were used as received. Purified water (18.3 MΩ) was supplied by a Milli-Q system from Millipore. Methods. The XR and GIXD experiments were performed using a Teflon trough (40 mL) with one movable barrier. DPPG was spread from a 1 mg/mL chloroform solution using a Hamilton microsyringe. After being spread, the monolayer was slowly compressed to 30 mN/m and maintained at that pressure throughout the remainder of the run. Addition and circulation of peptide and silicic acid solutions were accomplished using a peristaltic pump with inlet and outlet tubes submerged into each end of the trough. Circulation of one trough volume occurred in ∼10 min. The subphase was circulated for at least 30 min to ensure thorough mixing. All tubing and fittings were made of Teflon. The trough was maintained at 20 °C. Silicic acid was prepared just prior to injection into the subphase by dissolving sodium metasilicate in Millipore water (18.3 MΩ) and then adjusting the pH to 7.2 by addition of HCl. XR and GIXD were performed on the liquid surface spectrometer on beamline ID-9 at the Advanced Photon Source (CMC-CAT, Argonne National Laboratory) using a wavelength (λ) of 0.923 Å. Precautions were taken to prevent beam damage of the lipid monolayer during the XR and GIXD scans. First, the trough container was continuously purged with helium to minimize oxidative degradation of the organic monolayer. Second, the trough was moved by 1 mm in the horizontal plane, perpendicular to the incident beam, following each XR or GIXD scan. The reproducibility of multiple runs was excellent. In specular X-ray reflectivity, the ratio of the intensity reflected at the specular angle (θ) to the incident intensity is collected as a function of qz ) 4π sin θ/λ. Such a reflectivity curve is determined by the electron density profile normal to the interface. However, due to the loss of phase information in the intensity measurement and to the limited range of qz, the profile cannot be determined by direct inversion of the data, and a fitting procedure must be employed. In this work, fitting of the reflectivity data was performed using the GAFIT program based on the optical matrix method.20,21 GAFIT is available at www.ncnr.nist.gov. In the GAFIT program, the roughness parameter (r) is the full width at half-maximum ()2.35σ, where σ is the standard deviation) of a Gaussian distribution and in nearly all cases was (20) Russell, T. Mater. Sci. Rep. 1990, 5, 171–271. (21) Als-Nielsen, J.; Jacquemain, D.; Kjaer, K.; Leveiller, F.; Lahav, M.; Leiserowitz, L. Phys. Rep. 1994, 246, 251–313.
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Figure 1. Illustration of silicification induced by the membrane-bound peptide in the present system.
constrained in the fitting to be less than the smallest thickness (t) of the two adjacent layers. In fitting the data for the DPPG monolayer alone, the monolayer was described by two layers corresponding to the headgroups and the tails. The electron density within the tail layer was constrained to be consistent with the known area per molecule from the pressure-area isotherm. Upon introduction of the peptide, this constraint was removed to allow for the possible insertion of the peptide. In addition, to provide an adequate fit to the data, it was necessary to divide the lipid tail portion into two layers and to also include one additional layer below the lipid headgroups. After introduction of silicic acid, the complexity of the model profile was gradually increased in the following manner to achieve an adequate fit. First, one or two additional layers were included below the lipid headgroups to represent the silica, with the layers representing the lipid tails and headgroups remaining fixed at the values obtained prior to adding silicic acid. This alone was not sufficient to provide an adequate fit to the data. The electron density of the lipid head groups was then also allowed to vary in the fits, which was still not sufficient. However, upon allowing the electron density of the tail layers as well as the headgroups to vary, a good fit to the data was obtained. The total length of the lipid layer was also allowed to vary, but the fits consistently converged to the same length as that obtained prior to adding silicic acid. Dynamic light scattering measurements were performed with a Wyatt Dawn EOS (Wyatt Technologies, Santa Barbara, CA), and particle sizes were determined with QELS Batch software. The solutions were filtered with a 0.2 µm Anotop filter (Whatman, USA) prior to the measurements.
Results and Discussion A model of the DPPG/peptide/silica system consistent with the results is shown in Figure 1. Upon introduction of the peptide to the lipid monolayer at 1 mg/mL (0.4 mM), the peptide was observed to interact with the DPPG monolayer by several experimental techniques. The total surface area began to increase within minutes after circulation of the peptide and increased by a total of 18%. XR data for the DPPG monolayer (Figure 2a) display characteristic fringes resulting from the elevated electron density of the headgroups and the length of the aliphatic tails. The fitting parameters are given in Table 1. After the peptide was circulated, the fringes in the XR data decreased in magnitude and shifted slightly, indicating a substantial change in the characteristics of the monolayer film. The electron density profile after peptide adsorption (Figure 2b) indicates that the electron density of the lipid headgroup layer decreased, which is consistent with insertion of a substantial fraction of the peptide into the headgroups, as the peptide has a lower electron density than the DPPG headgroups. To obtain a good fit to the data, it was necessary to divide the lipid tail region into two layers. After peptide adsorption, the electron density of the lipid tailgroups increased in the region nearest the headgroups but decreased at the air interface. This is consistent with insertion of amino acid residues a short distance into the lipid tails, as the electron density of the peptide is greater than that of the lipid tails. Insertion into only the lower portion of the lipid tails accounts for the elevated
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Langmuir, Vol. 25, No. 1, 2009 307 Table 1. Fitting Parameters from Analysis of XR Data Fe/Fe,sub (×106 Å-2)
t (Å)
r (Å)
lipid tails lipid heads subphase
DPPG Only 0.89 1.30 1.00
15.8 9.7
8.2 9.4 4.8
lipid tails lipid tails lipid heads peptide subphase
With Peptide 0.83 0.97 1.16 1.05 1.00
8.9 7.2 11.7 7.1
8.4 5.4 6.9 5.9 4.6
layer
Figure 2. (a) X-ray reflectivity data for DPPG on the phosphate buffered subphase (O), after introduction and circulation of the peptide (9), and after subsequent dilution of the subphase (+). The data are plotted as R × qz4 to compensate for the qz-4 dependence of the Fresnel law and more clearly display the features in the data. A large change in reflectivity indicates insertion of the peptide into the lipid film. The effect persists after subphase dilution, demonstrating that insertion of the peptide is irreversible with respect to bulk peptide concentration on the experimental time scale. (b) Fitted electron density profiles for DPPG alone (- - -) and after circulation of the peptide (-) revealing insertion of the peptide into the lipid monolayer. A portion of the peptide is exposed to the subphase below the lipid headgroups.
electron density in that region and also the decreased packing density of the tails at the air-tail interface. Finally, a layer of increased electron density between the headgroups and the subphase was essential to obtain a satisfactory fit, indicating that a portion of the inserted peptide extends 10 Å ( 3 Å into the solution below the lipid headgroups. Importantly, the very limited extent of this layer rules out the adsorption of peptide aggregates. This is the case despite the fact that dynamic light scattering measurements have shown that the present peptide forms aggregates of 30-400 Å in bulk solution at the present conditions, similar to previous reports for R5 and RRIL-containing variants
lipid tails lipid tails lipid heads silica 1 silica 2 subphase
With Silicic Acid - 48 min 0.80 8.9 0.98 7.3 1.18 12.7 1.22 10.4 1.27 25.0 1.0
8.1 4.2 4.4 6.9 10.0 25.0
lipid tails lipid tails lipid heads silica subphase
With Silicic Acid - Final 0.82 9.0 0.89 7.3 1.15 12.7 1.28 11.0 1.0
8.1 4.2 4.4 10.0 10.0
at higher concentrations of peptide (2 mM) and salt (100 mM phosphate buffer).14 The results in Figure 2b suggest that only monomeric peptide adsorbs to the surface. Finally, insertion of the peptide into the lipid membrane was found to be irreversible with respect to the bulk peptide concentration over several hours (Figure 2a). These findings are reasonable based on the peptide sequence. The peptide is highly charged at pH 7 (+8), consistent with strong interactions with the negatively charged DPPG headgroups. The positively charged residues (K and R) are clustered near the C-terminus. The majority of the residues are hydrophilic (16 of 23 residues), and the hydrophobic residues are distributed throughout the sequence, with two tyrosines and a glycine on the N-terminus and two IL pairs near the C-terminus. Thus, we would expect insertion of small portions of the peptide only short distances into the lipid tails. An alternative explanation for the change in the electron density profile upon peptide adsorption (Figure 2a) is that the lipids stagger such that a fraction of the lipid molecules are displaced roughly 10 Å into the subphase. Such a substantial staggering of the lipids would have the effect of decreasing and smearing out the electron density of the lipid headgroups and also of increasing the roughness of the tail layer. A small degree of staggering is likely as the peak in the profile is more rounded after addition of the peptide. However, staggering of the lipids does not account for the observed increase in surface area, nor does it account for the increased electron density in the portion of the tail region nearest the headgroups. Staggering of the lipids by 10 Å was deemed improbable, as it would expose a substantial portion of the hydrophobic tails to a hydrophilic environment. Furthermore, if the elevated electron density below the original headgroup position were due to staggering of the lipids rather than adsorbed peptide, then the adsorbed peptide itself would have no contribution to the electron density profile. This result would require a very low level of peptide adsorption, which is in direct conflict with the substantial increase in surface area observed. The effect of peptide insertion into the lipid membrane is also evident in GIXD scans (Figure 3). At 30 mN/m, DPPG is in the gel phase and the aliphatic chains order into a distorted hexagonal
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Figure 4. X-ray reflectivity scans as a function of time after introducing silicic acid (SA) into the subphase. The curves are shifted along the y-axis for clarity. The times after injection of silicic acid are indicated on the plot.
Figure 3. GIXD scans showing the ordered packing of the DPPG tails prior to peptide adsorption and the absence of ordered packing after adsorption of the peptide. This further demonstrates insertion of the peptide into the lipid tails.
lattice, resulting in two closely spaced diffraction peaks (Figure 2a).22 After the peptide was introduced, the diffraction peaks were no longer present (Figure 2b). This indicates that, rather than forming domains within the lipid membrane, the inserted peptide mixed with the lipids and disrupted the ordering of the lipid tails. Formation of peptide domains within the membrane would have resulted in only a small decrease in the magnitude of the peaks in proportion to the area fraction of the peptide domains (in this case, e18% as indicated by the increase in surface area). It is not possible to determine from the GIXD data alone whether the peptide inserts into the headgroups only or also inserts into the hydrophobic tail region. Past work has shown that disruption of the ordered packing of the tails can result in either case. In a prior GIXD study involving diphtheria toxin (DT) binding to monolayers of DPPG, complete loss of the Bragg peaks for DPPG occurred only for conditions (sufficiently high concentration and low pH) that led to insertion into the lipid tails.23 At lower DT concentrations, the protein bound to the headgroups but did not insert into the tails and had no effect on the DPPG gel phase. On the other hand, another GIXD study involving peptides adsorbing to 1,2-disterylglycero-3-triethyleneoxideiminodiacetic acid showed that disruption of the gel phase can occur in the absence of insertion into the lipid tail layer.24 We note that staggering of the lipids would not be expected to result in the complete loss of the Bragg peak. (22) Neville, F.; Cahuzac, M.; Konovalov, O.; Ishitsuka, Y.; Lee, K. Y.; Kuzmenko, I.; Kale, G. M.; Gidalevitz, D. Biophys. J. 2006, 90, 1275–1287. (23) Kent, M. S.; Yim, H.; Murton, J. K.; Satija, S.; Majewski, J.; Kuzmenko, I. Biophys. J. 2008, 94, 2115–2127. (24) Yim, H.; Kent, M. S.; Sasaki, D. Y.; Polizzotti, B. D.; Kiick, K. L.; Satija, S. Phys. ReV. Lett. 2006, 96, 198101–198104.
Silicic acid was then injected into the subphase underneath the lipid-peptide monolayer and circulated for 30 min (three trough volumes) to determine if silica precipitation would occur. After silicic acid was introduced, new fringes appeared indicating the formation of a new layer at the interface. These fringes increased in magnitude as a function of time and shifted to higher qz (Figure 4). These changes continued for roughly 5 h, after which little further change was detected. The best-fit electron density profile (Figure 5) indicates a layer of silica below the lipid membrane, presumably stimulated by the exposed portion of the peptide. The data indicate that very little silica formation occurred within the lipid headgroup or tail regions. Initially, the layer of silica was ∼40 Å thick with a maximum density of 1.30 g/cm3. Over time, the thickness of the layer gradually decreased but the maximum density remained roughly constant. In the final state, the most dense portion of the layer was approximately 15 Å thick. The density corresponds to 56% of that of natural cristobalite (2.32 g/cm3).24 It is important to note that this density is an in-plane averaged value. For example, the layer could be heterogeneous, with 23% of the area consisting of silica domains at 2.32 g/cm3 and 77% water, or the layer might be homogeneous with a uniform density of 1.30 g/cm3. A small decrease in area (∼5%) occurred gradually after circulation of silicic acid that coincided with the changes in the reflectivity data following the scan at 48 min. The area stabilized to a constant value after 5 h, correlated with the onset of nearly constant reflectivity. This decrease in area most likely indicates removal of some peptide from the surface layer as the silica formed. The electron density profile (Figure 5b) shows a loss of electron density from the lipid tail region as compared to the previous profile (Figure 5a), providing additional evidence of peptide loss from the monolayer over this time period. The thinning of the silica layer (Figure 5), observed simultaneously with the decrease in surface area and the decrease in electron density in the lipid tail region, may correspond to nanoparticles of silica that bud off from the developing layer, thereby removing some peptide from the monolayer in the process. We note that the thickness of the silica layer continuously decreased with time from that of the initial measurement. In particular, no
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Figure 6. X-ray reflectivity for DPPG on phosphate buffered subphase (b), and scans initiated 30 min (0) and 90 min (9) after introduction of silicic acid. The lack of change indicates that no silica forms in the absence of the peptide.
Figure 5. Time evolution of the nanosilica structure. Best-fit electron density profiles are shown before (- - -) and after (-) introducing silicic acid. The silica layer becomes thinner with time.
oscillations in thickness with time were observed, as would be expected for a budding and regrowth process. Thus, no additional deposition of silica occurred after the initial interfacial layer had formed. GIXD scans were collected over the range of qxy from 0.95 to 1.66 Å-1 after introducing silicic acid. The absence of peaks indicates that the interfacial silica is amorphous, consistent with that fact that the silica found in diatoms is amorphous.25 Finally, we note that no silica formation was observed at the DPPG/solution interface in the absence of the peptide (Figure 6). This demonstrates that the formation of interfacial silica (Figure 5) is catalyzed by the peptide. The fact that silica formation was catalyzed by an adsorbed peptide layer of very limited extent, essentially monomeric in dimension normal to the membrane, is significant in light of previously reported behavior for similar peptides in solution. Knecht and Wright reported a strong correlation between the RRIL sequence, peptide aggregation, and silica-precipitating activity for R5 and variants generated by site-directed mutagen(25) Sumper, M. Science 2002, 295, 2430–2433.
esis.14 Peptides containing RRIL formed aggregates of 60-450 nm and exhibited strong silica-forming activity, whereas most peptides lacking RRIL did not form aggregates and had much more limited silica-precipitating activity. They concluded that RRIL serves to promote peptide aggregation into active silicaprecipitating assemblies. The present work shows that an RRILcontaining R5-like peptide associated with lipid membranes catalyzes silica formation without forming large aggregates. We note that a limited degree of in-plane association cannot be ruled out on the basis of the present data. However, as mentioned above, the complete loss of the Bragg peak upon peptide adsorption rather than simply a decrease in proportion to the fractional increase in area rules out the formation of large peptide aggregates and suggests mixing of the peptide with the lipids on a molecular scale. The adsorbed amount of the peptide and the amount of silica formed at the interface can, in principle, be determined by integrating the electron density profiles. Integrating the portion of the profile due to silica (Figure 5a) results in 0.19 molecules of SiO2/Å2. In the case of the peptide, the results depend upon the fraction of the peptide that dangles below the lipid headgroups, which is unknown. For the profile with the adsorbed peptide but without silicic acid (Figure 2b), integrating the excess electron density below the headgroups and assuming that that portion accounts for one-fourth of the peptide while the rest of the peptide is inserted into the lipid headgroups and tails results in an estimate of 1.8 × 10-3 peptide molecules/Å2. On the other hand, if the portion below the headgroups corresponds to the entire peptide, the surface density estimate is 4.7 × 10-4 peptide molecules/Å2. The amount of silica formed per adsorbed peptide is therefore estimated to range from 105 to 420 mol of SiO2/mol of peptide in Figure 5a and from 30 to 120 mol of SiO2/mol of peptide in Figure 5b. Because the XR data indicate that a substantial fraction of the peptide is inserted, we believe that the lower values are more accurate estimates. For comparison, the activity of silaffin 1A in solution was reported previously to be ∼87 mol of SiO2/mol of peptide in one study12 and 12 mol of SiO2/mol of peptide in another.10 Using a modified molybdate assay with a 5 min reaction time,18 the activity of the current
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silaffin peptide in solution was found to be slightly greater than that of R5 (∼1.63 mol of SiO2/mol of peptide · min for the present peptide as compared to 1.15 mol of SiO2/mol of peptide · min for R5, the latter being in good agreement with previously reported data14). However, because the aggregation state of the peptide is different in solution and at the membrane, the activity is likely to differ in the two cases as well. A detailed comparison of the silica-forming activities of the present peptide, R5, and a peptide with the same sequence as R5 but lacking the KRRIL group, both in solution and adsorbed to membranes, will be described in a future report. It is noted that the length scale of the silica layer deposited at the lipid interface is much smaller than the diameter of silica particles found in the frustule of diatoms (∼40 nm).5 The growth of interfacial silica in this work (Figure 5) was limited to only a few nanometers. In additional studies with the present peptide at higher silicic acid concentrations, silica growth remained limited to e10 nm. The reduced length scale relative to that observed in the frustule of diatoms may be due to the post-translational modifications of R5 in silaffins, differences in the aggregation states of the peptides, and the difference in the amino acid sequence between the silaffin peptide used here and R5.
Conclusions This study shows that the use of planar lipid membranes combined with analysis by grazing incidence X-ray scattering methods can provide fine structural details of the silicification process at the lipid interface and provides the first direct insight into this complex interfacial process. We have proposed a hypothesis describing this phenomenon (Figure 1) on the basis of the results. The results indicate that the aggregation state of the peptide at the membrane is much less than that in solution, with a thickness of only 10 Å extending below the lipid headgroups as compared to peptide aggregates in solution with diameters
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ranging up to 400 Å. The data indicate that the peptide mixes with the lipid monolayer, disrupting the gel phase. A portion of the peptide remains exposed to the solution and acts as a catalyst for the deposition of silica beneath the lipid membrane. The initial deposition (40 Å) of silica occurs very rapidly, followed by a gradual decrease in the thickness of the layer. This decrease in thickness may be due to gradual desorption of peptide induced by silica formation. The very high peptide concentration (1 mg/ mL) and the fact that the peptide was removed from the subphase prior to introduction of silicic acid may have contributed in that regard. Silica growth was limited to several nanometers. The effects of the precise amino acid sequence and the concentrations of the peptide and silicic acid on the kinetics of silica growth and the structure of the final interfacial layer will be described in a future report. This work raises the possibility that controlling lipid membrane composition and subsequent interactions with catalytic peptides may be a useful mechanism to direct silica growth in synthetic and biomimetic systems. In particular, phase separation of lipid membrane components could be used to create domains that promote or inhibit adsorption of silaffin peptides and silica formation, creating novel nanostructured films with applications in coatings, catalysis, and separation science. Acknowledgment. Sandia is a multiprogram laboratory operated by Sandia Corp., a Lockheed Martin Co., for the U.S. Department of Energy under contract DE-AC04-94AL85000. Work at the CMC beamlines is supported in part by the Office of Basic Energy Sciences of the U.S. Department of Energy and by the National Science Foundation Division of Materials Research. Use of the Advanced Photon Source is supported by the Office of Basic Energy Sciences of the U.S. Department of Energy under Contract No. W-31-109-Eng-38. LA801794E
