Article pubs.acs.org/crystal
Growth of Ultrastable Protein−Silica Composite Crystals Jose A. Gavira,* Alexander E. S. Van Driessche, and Juan-Ma Garcia-Ruiz Laboratorio de Estudios Cristalográficos, IACT (CSIC-UGR), Av. La Palmeras 4, E18100, Armilla, Granada, Spain S Supporting Information *
ABSTRACT: Protein crystals were obtained in a wide range of silica gel concentrations, 2.0−22.0% (v/v), using the counter-diffusion technique. The protein crystal lattice incorporates silica fibers during their growth, making the crystal appear optically translucent while maintaining the diffraction quality. The effect of the silica fibers on the nucleation and growth morphology is discussed, and the amount of incorporated silica matrix is quantified. The practical implications of the presence of a high hygroscope phase on the crystal properties are discusse, and the improvement of the mechanical properties and stability of the crystals is shown. These reinforced protein crystals, able to include large amounts of silica, open a new range of possibilities for the characterization of protein crystals and the application in the biotechnological industry. protein single crystals of up to 30 mm.16 The protein crystal lattice is able to incorporate large amounts of silica while still maintaining its short-range crystallographic order. Additionally, the mechanical properties and the stability of the crystals against dehydration are improved by the incorporation of a hydrophilic silica polymeric network to such an extend that it was possible to record full diffraction data sets (with a resolution better than 1.5 Å) of a crystal protected only by a layer of mineral oil.16 Besides the crystallographic interest in obtaining reinforced protein crystals able to maintain their structural integrity during their manipulation and to resist dehydration, these “hybrid” crystals open a new door for the measurement of physical properties of large biological macromolecules as well as for their technological applications, i.e., as nanocomposite scaffolds.17 To further investigate the possibilities of growing reinforced protein crystals, we have selected three model systems and studied the silica−protein interactions in solution and the influence of silica gel on the nucleation and growth and the physical properties of reinforced crystals.
1. INTRODUCTION Protein crystals can be considered as soft and fragile material due to the low reticular energy and large solvent content, 30− 80% (v/v).1 Hence, protein crystals are sensitive to any type of stress such as mechanical shocks, osmotic pressure, drying, etc. As a direct consequence, this fragility plays a significant role in the final quality of the X-ray diffraction data. On the other hand, the solution contained in protein crystals maintains the same characteristics as the mother solution from which the crystals grew,2 allowing the free diffusion of small molecules that can interact/react with the protein just as in free solution.3 Different strategies have been applied to improve the stability of protein crystals mainly for data collection4 such as crosslinking with glutaraldehyde5 or cryo-crystallography.6 Alternatively, in situ X-ray diffraction focuses on minimizing crystals manipulation. This approach is fully exploited by the capillary counter-diffusion method in which crystals are obtained inside X-ray transparent capillaries.7 The counter-diffusion method explores a wide range of supersaturation conditions in a single experiment while providing a stable environment to introduce or remove additives.8 Although protein crystals obtained in capillaries media avoid their manipulation, the maximum attainable crystal size is limited by the capillary diameter (0.1−0.3 μm). The most frequently used techniques for characterization of optical, magnetic, mechanical, and other physical crystal properties require stable and large and/or high quality crystals. Stability and size are therefore important limitations to explore the possibilities of macromolecular crystals for technological purposes, e.g., computer memory,9,10 drug administration,11,12 enzymatic activity,13−15 etc. The combination of silica gel and the counter-diffusion technique permits researchers to obtain © XXXX American Chemical Society
2. MATERIALS AND METHODS 2.1. Proteins and Chemicals. Three times recrystallized hen egg white lysozyme (cat. no. L-6876) and thaumatin from the arils of the African rub Thaumatococcus daniellii (cat. no. T-7638) were purchased from Sigma (St. Louis, MO) as lyophilized powders and were used as received. Fresh stock solutions of 200 mg/mL were buffered in 50 mM sodium acetate (pH 4.5) and sodium phosphate 100 mM (pH 6.5), Received: February 8, 2013 Revised: May 7, 2013
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respectively. The stocks solutions were filtered (0.45 μm, Millipore, Millex-HV13), and the concentration was determined spectrophotometrically measuring the absorbance at 280 nm using 2.66 mL·mg−1 cm−1 for lysozyme and 1.22 mL·mg−1 cm−1 for thaumatin as extinction coefficients. Ferritin was obtained as a concentrated solution from Sigma (code F-4503) and diluted to the desired concentration with sodium acetate 50 mM pH 5.5. 2.2. Silica Gel. Silica sols were prepared with tetramethoxysilane (TMOS, C4H12O4Si, MW 152.22 g/mol, Fluka Chemie, cat. no. 87680) by mixing the appropriate volume of TMOS with the crystallization buffer in continuous agitation until a homogeneous solution was obtained. The monomeric silica stocks solution (sol) was prepared at twice the desire final concentration. The gelling time of silica-sols strongly depends on the silica-sols concentration and pH. When the silica concentration is increased, the gelling time decreases and becomes minimum at pH 7.0.18 Hence, the highest obtainable silica gel concentration is restricted by the gelling time and needs to be prepared extemporaneously. In this work, we prepared experiments up to 22% silica gel final concentration (44% stock solutions). At these high silica concentrations, up to 30 min of continuous vigorous agitation is needed to obtain a homogeneous solution. 2.3. Crystallization and Dissolution. In this study, crystallization experiments were done using a three-chamber counter-diffusion geometry.19 A glass tube was used as the growth reactor (length ≈ 90 mm and inner diameter ≈ 4.0 mm). Equal volumes of protein solution and fresh silica sol were mixed and immediately transferred into a glass tube (40 mm of protein chamber). Once the sol-protein was gelled, a 5 mm physical-buffer (4% (v/v) buffered silica sol) was poured gently on top. This physical buffer layer serves to smooth the high initial supersaturation values that could be reached if a concentrated salt solution enters directly in contact with the gelled protein. The crystallization tubes were stored vertically overnight. During the last step, the precipitant solution was added on top of the physical buffer layer to complete the 40 mm precipitant chamber. Experiments were stored at room temperature. When ferritin was mixed directly with the silicic acid sol, flocculation occurred. To avoid this a two-step preparation protocol20 was used in which the protein solution was first loaded in the gel by diffusion after the gel had set. Second, the protein solution was removed and replaced by the precipitant solution. We evaluated nucleation by extracting the silica cylinder and counting the total number of crystals. Cylindrical silica gels, containing protein crystals, were extracted from the tube by increasing the pressure at the precipitant end using a syringe or a cotton-stick. Individual crystals were separated from the gel using a paintbrush and crystal manipulation tools (Hampton Research, Laguna Niguel, CA). Crystal dissolution experiments were done by transferring selected crystals to a glass Petri dish under an Olympus binocular microscope equipped with a camera. The mother solution was then undersaturated by adding an equal amount of distilled water (i.e., protein and salt concentration were reduced by half). 2.4. Electron Microscopy. High-resolution micrographs were obtained with a ZEISS DSM 950 electron-scanning microscope working at 4−7 kV. The cylindrical silica gel containing lysozyme crystals was cut into 1 cm pieces, deposited over an aluminum disk of 6 cm diameter sample-holder, and immediately immersed in liquid nitrogen. The disk was transferred to a vacuum chamber precooled at 193.5 K and equilibrated with argon. While pressure was kept constant at 10−6 atm, the temperature was increased from 193.5 K to room temperature over 48 h to sublimate the water contained in the sample. The 1 cm dry samples were cut in small pieces using a scalpel under a binocular microscope. When possible, individual crystals were carefully fractured to display the inner core. Each piece was fixed on a two-face stick carbon disk glued on a standard aluminum SEM pin stub. Each aluminum support was individually coated with gold using a POLARON E-5000. During the whole process, the samples were kept in a dry environment. A detailed description of the preparation protocol is described elsewhere.19 2.5. X-ray Characterization. Crystal quality was evaluated by Xray diffraction. Crystallographic analysis was performed on a home
source as well as synchrotron X-ray radiation source. Protein crystals were mounted in glass capillaries sealed with bee wax for room temperature (RT) data collection (lysozyme) and in loops, after equilibration with 15% (v/v) glycerol, for 110 K data collection (thaumatin). The Cuα rotating anode home source generator was operated at 50 kV and 100 mA and diffraction images were recorder in a MAR detector. A partial data set was also collected at beamline BM16 of the ESRF synchrotron light source using 0.976 Å wavelength. Data were indexed, integrated, and scaled using the HKL2000 package.21
3. RESULTS AND DISCUSSION 3.1. Silica−Protein Interactions in Solution. Protein interactions with a suspension of colloidal silica particles happen mainly through hydrogen bonds between silica hydroxyls groups and electron donor atoms (oxygen, nitrogen, ether and amide groups) of protein residues.18 Protein−silica interactions can provoke the precipitation of proteins and other organic substances due to the formation of hydrogen bonds with the silica particles, even in the absence of salt.22 This behavior is frequently observed when the protein solution is mixed with a silica sol prepared by neutralization of sodium metasilicate but occurs rarely when the silica-sol is prepared by the monomerization of alkoxides (TMOS or TEOS). When using the counter-diffusion technique, the buffered protein solution is mixed with the buffered TMOS/TEOS sol, which avoids flocculation, although interaction of the protein molecules with the monomers of silicic acid occurs through the hydroxyl groups at the early stages of mixing. These interactions accelerate the polymerization of the silica particles, and thus reduce the gelling time. In Figure 1 the reduction of
Figure 1. Gel point as a function of TMOS concentration. Squares correspond to gels prepared in 50 mM sodium acetate pH 4.5 and circles correspond to gels prepared in buffered lysozyme at 100 mg/ mL.
the gel-point (time at which the gel stops to flow), due to the interaction of silica particles with lysozyme molecules, is shown. This tendency was also observed for thaumatin and insulin.18 In this work, we found that flocculation occurs when model protein ferritin was mixed with both TMOS and TEOS. This behavior can be attributed to the high content of salt, 0.15 M NaCl, present in the commercial ferritin solution because in the presence of salt stronger interactions between the silica particles and protein molecules are established.18 Protein− silica flocculation can be avoided using a two-step sample preparation protocol.23 Following this protocol, a ferritin solution was allowed to diffuse for three months into silica B
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polymerization of TMOS remains in the system. Taking into account the specific gravity of methanol 0.787 g/cm3 and knowing that a maximum of 4 mol can be produced per mole of TMOS, 1.1% (v/v) of methanol is generated by increasing the gel concentration by 1% (v/v). (ii) During the condensation of silica-sols protein molecules can also be permanently entrapped by the silica particles and/or can be strongly interacting with the remaining hydroxyl group covering the silica surface, and thus, temporally, the effective protein concentration in solution will be lowered. Currently, it is not possible to differentiate the contribution of both mechanism, the protein−gel interaction (permanent and transitory), and the effect of methanol on the solubility of lysozyme, on the reduction of the nucleation density. As expected, in the silica gel concentration range of 2.0− 12.0%, the reduction of the nucleation rate is accompanied by an increase of the crystal size when compared with similar experiments in agarose or in gel-free media. Lysozyme crystals with a volume of up to 30 mm3, with high diffraction quality, have been obtained by counter-diffusion methods using silica gels.16 This can be explained taking into account that once nucleation takes place protein concentration is depleted in the vicinity of the nucleus, which will trigger a controlled desorption of silica-absorbed protein molecules and favor the steady growth of protein crystals. Above 12.0% (v/v), increasing gel concentrations provoke the average crystal size to decrease due to increasing protein solubility and entrapped protein molecules. This reduction of the crystal size is also accompanied by an important morphological transition that is treated at the end of this section. When model protein thaumatin was crystallized in the presence of silica gel also an increase in crystal size and a reduction of the nucleation is observed, but no significant correlation between the nucleation density and silica concentration was observed. Although it is worth mentioning that for high silica concentrations ≥15% secondary nucleation of thaumatin crystals occurred in the bulk of the gel and not on the edge of the primary formed thaumatin crystals, something that can be observed for gel-free solutions or gels of low to medium silica concentration (≤15%, Figure 3). If we assume that in the gel-free case these nucleation events occur at surface sites due to an accumulation of defects, which lowers the surface tensions and consequently lowers the nucleation barrier,
gels. A dark-colored precipitate was observed at the interface with the protein solution that did not redissolved over time. Despite this, the experiment was run, and typical dendritic crystals of ferritin were obtained. 3.2. Influence of Silica Gel on Nucleation and Growth of Protein Crystals. One of the most striking observations during the crystallization of lysozyme in silica gels is the reduction of the nucleation density20,24 Figure 2 shows the
Figure 2. (A) Microscope image showing the variation of the nucleation density of lysozyme as a function of TMOS concentration from 8% to 18% (v/v). The diameter of the tubes is 4 mm. (B) Graphic representation showing the total number of crystal as a function of gel concentration (full symbols) and the increment of the protein concentration in solution measured at equilibrium (open symbols) spectrophotometrically at 280 nm. The solubility of lysozyme at the NaCl equilibrium concentration in a gel-free system is shown with an open square.
reduction of the nucleation density of lysozyme crystals as a function of gel concentration. Two plausible mechanism are considered to explain this observation: (i) In the presence of methanol the solubility of lysozyme slightly increases.20 This is clearly observed when the equilibrium concentration of lysozyme is plotted as a function of the initial gel concentration (Figure 2). The total amount of methanol produced during the
Figure 3. (A) Nucleation density of thaumatin crystals in silica gels of different concentrations. (B) Secondary nucleation on the edges of thaumatin crystals growing in a low concentration silica gel (5%). (C) Thaumatin crystals growing in a high concentration silica gel (20%) showing no secondary nucleation on the rounded crystal edges. C
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Figure 4. (A) Schematic representation of the protein crystal growth and equilibrium morphology as a function of silica gel concentration. Wide field images of lysozyme (B) and thaumatin (C) crystals as a function of silica gel concentration.
faces is observed. At higher gel concentrations (>15.0%), rounding of the apexes of the (110) faces also occurs. This eventually leads to ellipsoidal crystal habits in which the (101) and (110) faces could not be distinguished anymore. Thaumatin crystals showed a more abrupt transformation of the crystal shape with increasing gel concentration. The first changes in growth morphology occur for crystals grown in 10.0% silica gels. At this gel concentration, the apexes of the (101) faces become rounded. Although this rounding is more pronounced along the [001] direction, it is also visible along the four apexes of the (110) plane (Figure 4). For silica gel concentrations >10%, these features become more evident and eventually lead to elongated ellipsoidal shaped crystals where individual faces cannot be distinguished, as in the case of lysozyme. In the concentration range of 4−15%, faint central depressions on the faces were occasionally observed. To tentatively explain these observations, two different mechanisms are considered, one being responsible for the face instability, and a second one accounting for the evanishing of externally recognizable crystallographic faces (i.e., loss of a wellfaceted crystal habit). The appearance of a depression in the central part of the crystal faces for both proteins, lysozyme and
we can assume that thaumatin crystals with rounded edges, grown at high gel concentrations, are free from these defects. Although we also have to consider that the depletion zone around growing crystals will be more pronounced and maybe the critical supersaturation for secondary nucleation cannot be reached anymore. A second, remarkable feature of protein crystals grown in silica gel is that the growth and “equilibrium” morphology of the crystals are strongly controlled by the gel concentration, as shown in Figure 4. For both model proteins, lysozyme and thaumatin, we observed that increasing silica concentration the crystal shape evolves from well-faceted crystals to rounded ellipsoidal-shaped crystals without recognizable faces or apexes (Figure 4). In the case of lysozyme, a very gradual transition from wellfaceted crystals to rounded ellipsoidal crystals is observed for the silica gel concentration range from 2.0 to 22.0%. At lower gel concentrations (4.0−10.0%), face instability appears. Shallow depressions are observed in the center of the (110) and (101) faces in the concentration range of 4.0−10.0%. At medium gel concentrations (10.0−15.0%), the face instabilities gradually disappear, and rounding of the apexes of the (101) D
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thaumatin, is most likely due to the “Berg effect”.25−27 This occurs when the effective supersaturation is higher at the corners of the crystals compared to the center of the crystal faces; this is a direct consequence of the spherical shape of the depletion zone around a growing faceted crystal and can lead to face instability.27,28 Taking this into account, it is reasonable to assume that in gelled solutions it is expected to observe the Berg effect, especially at high supersaturation (close to the physical plug/protein chamber interface). Sleutel and coworkers have described stronger polyhedral instability when studying the growth of glucose isomerase crystals grown in gelled solutions compared to solution grown crystals.26 Nevertheless, the central depressions observed in our work are only of transitory nature, i.e., kinetic effect, and eventually disappeared when the supersaturation along the tube tends to zero. At higher gel concentrations (>10% for lysozyme and >15% for thaumatin), the Berg effect becomes less pronounced, and a gradual transition to rounded crystals is observed. The rounding of crystal corners and apexes is not of a transitory nature because when the supersaturation inside the tube tends to zero the round shape is maintained. Hence, it is not a consequence of the Berg effect and is thus most likely the result of a thermodynamic effect such as a decrease in the effective surface free energy (αeff) due to incorporation of silica fibers by the growing protein crystal faces at higher gel concentrations. A previous study with agarose gel showed that the incorporation of gel fibers lowers the effective surface free energy of the crystal surface.28 In this study, only low agarose gel concentrations were used (
