Controlled Crystallization of Macromolecules using Patterned

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Controlled Crystallization of Macromolecules using Patterned Substrates in a Sandwiched Plate Geometry Anindita Sengupta Ghatak and Animangsu Ghatak* Department of Chemical Engineering, Indian Institute of Technology, Kanpur 208016, UP, India ABSTRACT: Crystallization of macromolecules such as proteins and peptides is known to be influenced by the topographical and chemical heterogeneity of the substrate. However, controlling the nucleation and the growth of crystal on such surfaces has been an issue. Here, we present systematic experiments carried out on hydrophilic elastomeric substrates topographically patterned by forming stretch induced surface wrinkles; the distance between the wrinkles, importantly the density of occurrence of defects between the wrinkles, is systematically varied. Furthermore, to maximize the effect of the substrates, the crystallization experiment is carried out between two such parallel substrates, the gap between which is maintained by using spacers. This process results in very controlled evaporation of the solvent. Experiments with two different model proteins: hen egg-white lysozyme and Thaumatin from Thaoumatococcus daniellii show that on surfaces with uniformly spaced wrinkles the crystals nucleate extensively but with insignificant growth. However, when a small number of defects are introduced into the patterns, fewer crystals nucleate, which grow to form large crystals. With further increase in the defect density, extent of nucleation increases again, but with decrease in the crystal growth. Thus, the crystal size attains maxima at an intermediate wavelength of the wrinkles and the defect density.

1. INTRODUCTION Understanding crystal structure of proteins is important for diverse pharmaceutical and medical applications, e.g., disease diagnoses,1 drug design,2 drug delivery,3 development of genetically modified crops,4 biological pest control,5 controlling oil spillage,6 and even green-energy generation.7 Central to all these applications is the process of nucleation of protein macromolecules and their growth to a crystal. Several process variables are known to affect the nucleation and growth of crystals, e.g., rate of evaporation of water, concentration of proteins in the buffer solution, chemical nature of precipitants and additives, temperature of the surrounding environment, pH of the buffer solution, and even the substrate topography. Interplay of all these parameters often results in a complex situation at which growing diffraction quality crystals of many proteins becomes almost impossible to accomplish. For example, in the conventional vapor diffusion method, crystals are grown from supersaturated aqueous solution on a silanized glass/plastic coverslip wherein high concentration of the protein needs to be used.8 The drawback of this process is that here a large number of tiny crystals are formed because of excessive nucleation. Another difficulty is the precipitation of protein molecules, which occurs even before overcoming the free energy barrier for nucleation of the stable clusters.9 Getting around such problems has no panacea and in fact it demands several cycles of trial and error with different combinations of protein concentrations, buffers, precipitants, additives, and so on. In this context, use of chemically and/or physically modified surfaces for heterogeneous nucleation has been a step forward toward achieving the directed crystallization of proteins.10 For example, the potential energy barrier for nucleation of proteins was found to decrease on different mineral substrates11 which have been used as epitaxial nucleants for nucleating common proteins like canavalin, concavalin, beef liver catalase, and hen egg lysozyme. Similarly, several other substrates, silanized mica surfaces,12,13 silanized polysterine wells,14 and polymeric film surfaces,15,16 have r 2011 American Chemical Society

been found to promote nonspecific electrostatic attraction between the exposed charged residues of protein and the surfaces. These surfaces can reduce the induction time for crystallization, thereby increasing the crystal size. While these are the examples of chemically heterogeneous surfaces, several others have been used for which substrate topography engenders the heterogeneity. For example, a variety of porous substrates such as porous silicon,17 mesoporous bioactive gel-glass,18 porous glass surfaces19 with pore sizes (2
10 nm), comparable to that of protein molecules have been used as the heterogeneous nucleants. However no systematic experiment has been done by varying the surface topography of a single substrate. In this report, we present a systematic way to control the nucleation and growth of protein crystals by physically texturing the substrate. We have used poly(dimethylsiloxane) (PDMS) elastomeric substrates which we have patterned to form one-dimensional near parallel undulations having wavelengths in the range of 200
800 nm. In addition, we have inserted defects in the form of kinks, in limited numbers, within these undulations. We have shown that presence of these defects significantly influences the crystal sizes which maximize at an intermediate density of occurrence of the defects. We have crystallized two model proteins, lysozyme and thaumatin, to demonstrate the generality of this process.

’ EXPERIMENTAL SECTION Figure 1a depicts the schematic of the crystallization setup which is different from the conventional hanging drop.8 Here Special Issue: Ananth Issue Received: December 30, 2010 Accepted: March 31, 2011 Revised: March 30, 2011 Published: April 19, 2011 12984

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Figure 1. (a) Schematic setup for crystallization experiment. AFM scans of patterned DVD (b), the first generation (c) and second generation (d) miniaturized patterns using DVD. AFM scans of patterns generated in stretched PDMS substrate: λ = 1.1 (e), 1.3 (f), and 1.4 (g). The plot (h) of stretching ratio vs defect density (λ ∼ F) and stretching ratio vs wavelength of pattern (λ ∼ η).

drops of protein
buffer
precipitant solution were placed between two substrates kept separated by keeping spacers of desired thickness, e.g., 120 μm, between them. Each drop of the liquid contained ∼4 μL of the solution, so that when confined between two parallel plates, it generated a liquid disk of initial average diameter 1.97 mm. Thus the exposed surface of the liquid through which evaporation could occur would be controlled by varying the gap between the substrates. The rate of evaporation was further controlled by suitably altering the hydrophobicity of the lower and the upper substrates.20 For example, the upper plate was silanized by coating it with self-assembled monolayer (SAM) of octadecyltrichlorosilane (OTS) molecules which rendered this surface hydrophobic; topographically textured poly(dimethylsiloxane) (PDMS) film attached to a rigid glass slide was used as the lower substrate. To texture the PDMS films two different procedures were followed: by successive miniaturization of lithographically patterned substrates21 and by the self-organization route of forming surface wrinkles.22 In either process the film was plasma oxidized before using it for crystallization experiments. Patterning by Miniaturization. Figure 1b
d show atomic force microscopy (AFM) images of several surface patterns prepared by miniaturization of the periodic stripes (periodicity, η = 686 nm, height, ε = 125 nm) of a commercially available digital versatile disk (DVD), which were regular without any structural defect. This pattern was transferred to the surface of a resorcinol-formaldehyde (RF) gel,21 which was slowly dried at controlled ambient conditions of 30 °C temperature and relative humidity of 75%. In this process, the periodicity of the stripes decreased to η = 420 nm and their heights decreased to ε = 25 nm. This pattern was transferred to thin films of PDMS by replica molding method. A similar sequence of steps for another cycle on these patterns further diminished the periodicity to 250 nm and the height to ε = 12 nm. Patterning by Surface Wrinkling. The second set of patterned substrates was prepared by stretching uniaxially thin strips

of PDMS films (length 25 mm, width 8 mm, thickness ∼300 μm) using a homemade gadget, followed by exposing it to oxygen plasma (plasma oxidized at a pressure of 0.05 Torr) for about four minutes, inside a plasma chamber. This process resulted in a thin crust of silicate layer on the PDMS surface, which turned hydrophilic. The stretched film was then released instantaneously which led to the appearance of wrinkles, because of the difference in stiffness of the thin crust and the elastic substrate. The periodicity and height of the wrinkles could be controlled by varying the initial extent of stretch λ = 1.1
1.5 (final length/ initial length). Figure 1e
g depicts the atomic force microscopy (AFM) images of the wrinkles generated on the PDMS surface with different initial extension ratio of the film, λ = 1.1, 1.3, and 1.4, respectively. Here, at small extension, e.g., 1.1 (Figure 1e), the wrinkles were uniformly separated with well-defined periodicity and height. However, with increase in λ these patterns turn increasingly nonuniform with the appearance of defects (Figure 1f
g) in the form of sharp kinks between two neighboring wrinkles. To calculate the defect density we considered a representative area of 5  5 μm2 in a typical AFM image of the patterned substrates and measured the number of defects that appear in that region per wave. The periodicity, η, of the wrinkles and the defect density, F, for different λ are summarized in Figure 1h which shows that periodicity as small as η = 225 nm is achieved at a moderate stretch of λ = 1.4. η decreases further with increase in λ but at the expense of increase in defect density F. Thus, η and F exhibit contrasting behavior while η decreases with λ, F increases, both showing linear dependence: η =
267λ þ 605.5, F = 1.72λ
1.786. The relation for F shows that the defects start appearing beyond the extension ratio, λ = 1.04. Furthermore, the histograms of the patterns show that when λ is small, e.g., λ = 1.1
1.3, the wrinkles appear with a single periodicity implying single mode of buckling of the crust. However, at large values of λ, Figure 1g, at least two different buckling modes appear very similar to fractal patterns observed in experiments with longer duration of plasma oxidation.23 12985

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Figure 2. Lysozyme and thaumatin crystals grown on patterned DVD (a,d), and the first generation (b,e) and second generation (c,f) miniaturized pattern substrates. The protein concentration was 20 mg/mL. (g) Comparison of the crystal sizes plotted in bar diagram.

’ RESULTS AND DISCUSSION The optical micrographs of crystals of two different proteins, lysozyme and thaumatin, obtained on different substrates are presented in Figures 2 and 3, which bring out contrasting details of nucleation and growth of crystals on these two different kinds of substrate. For example, the images in Figure 2a
g show that for miniaturized lithographic patterns (Figure1b
d) as the wavelength and height of the initial patterns, 686 and 125 nm, are decreased to 420 and 22 nm, i.e., by 38% and 82%, respectively,

the crystal size of lysozyme increases from 76 ( 4 to 84 þ 3 μm, i.e., by mere 11%. The crystals are however of uniform in size, which is measured by considering the longest dimension, d as show in Figure 2a. With further decrease in wavelength and height to 250 and 12 nm, i.e., by 40% and 45%, respectively, crystal size does not increase, rather it decreases by 12% to 76 ( 7 μm. Similar observations are made also for the thaumetin crystals and different other initial concentration of these proteins (Figure 2d
f). Thus, with surface patterns devoid of any defect, the crystal size alters 12986

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Figure 3. Lysozyme and Thaumatin crystals grown on stretched PDMS substrates: (a-b) protein concentration (c) = 10 mg/mL, for (c-f) c = 20 mg/mL and (g-h) c = 30 mg/mL. The two polymorphs (tetragonal and needle) of lysozyme are also indicated in the Figure. All pictures are taken at the same magnification.

Figure 4. Optical images of lysozyme (a) and thaumatin (b) crystals on PDMS substrates prepared by subjecting them to increasing initial stretching ratios λ. “d” indicates the length of crystal measured. All pictures were taken at the same magnification.

insignificantly in spite of large change in the wavelength and height of the patterns. The patterns of appearance of crystal, however, changed as the substrates changed with wrinkle patterns as in Figure 1e
g in which defects were present in different spatial densities. Figure 3a
b depicts the optical micrographs of lysozyme (initial concentration, c = 10 mg/mL) crystals obtained with two different initial extension ratios, λ = 1.1 and 1.4, which show remarkably different crystal sizes. For example, for λ = 1.1 the longest dimension of the crystals was 65.5 ( 0.8 μm and that for λ = 1.4 was 141.51 ( 0.01 μm. Similar results were obtained also for different other concentrations of the protein solution. For example, Figure 3c
d show that for c = 20 mg/mL and λ = 1.1 and 1.4, these crystals were of sizes 82.66 ( 1.65 and 159.02 ( 0.01 μm, respectively. We observe also the coappearance of two different crystal morphologies, tetragonal and needle-like, in the same sample. However, the two morphologies did not appear simultaneously but sequentially: with progress in evaporation of the liquid the tetragonal crystals of lysozyme appear first whole through the area of the liquid disk, which indicates that thermodynamically the tetragonal morphology is the most stable form. The needles appear at a later stage and more toward the periphery of the

shrinking liquid disk, which indicates that this morphology is possibly dictated more by pinning the contact line of evaporation. Therefore, the needle form appears to be kinetically favored. Unlike the tetragonal crystals, the needles grew in clusters radiating from a central zone. Such needles, however, did not appear if the PDMS surface was not oxidized, which proved that the chemical nature of the surface could control the crystal symmetry. In case of thaumatin, Figure 3e
f depicts the optical micrographs of crystals (initial concentration, c = 20 mg/mL) obtained with two different initial extension ratios, λ = 1.1 and 1.4. We observed that for λ = 1.1 the crystals nucleate more in number compared to λ = 1.4 for which the crystal dimension was significantly larger. This effect gets more pronounced as the protein concentration is further increased to c = 30 mg/mL as depicted by optical micrographs in Figure 3g
h, where the crystals were of sizes 106 ( 3 and 179 ( 1.2 μm, respectively. If the concentration of protein is increased beyond this limit (c > 50 mg/mL) the increased rate of nucleation tends to dominate the crystal growth and we see the appearance of a large number of tiny crystals, which do not grow to any significant extent. We summarize these observations in Figure 4a
b, in which we show a sequence of optical images of typical crystals on 12987

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Figure 5. Crystal sizes (d) plotted with wavelength (η) for lysozyme (a) and thaumatin (b). The two protein concentrations are indicated. The band indicates crystals grown from miniaturized pattern substrates. The inset pictures show the plot of estimated standard deviation of crystal size (Δd) with (λ
1).

PDMS substrates prepared by subjecting them to increasing initial stretching ratios λ. The figure shows that for both lysozyme (Figure 4a) and thaumatin (Figure 4b) the crystal sizes increase with increase in λ up to λ = 1.4, beyond which the crystal size decreases. Crystals as large as 180 μm can be obtained on these wrinkle-patterned substrates compared to the miniaturized lithographic patterned substrate and others.24 The question arises how exactly the stretching ratio λ influences the crystal size. As described in Figure 1h, variation in stretching ratio λ brings about two different types of changes in the topographical patterns of the substrate, e.g., wavelength and defect density, the coupled effect of which influences the crystal sizes. For example, as λ increases from 1.2 to 1.3, the wavelength η changes from 284 to 271 nm and the defect density F changes from 0.27 to 0.40; the height of the patterns remains nearly constant at 30
35 nm. Thus, change in wavelength by 4.6% and defect density by 48% result in a moderate increase in crystal size for lysozyme (initial concentration 10 mg/mL), from d = 69.5 to 75 μm, i.e., by 8%. However, the crystals get bigger as the wavelength η of the patterns decreases from 271 to 225 nm with increase in λ from 1.3 to 1.4; at this the defect density F increases from 0.4 to 0.65. This decrease in η by 17% and increase in F of 62% results in increase in crystal size to 142 μm, i.e., by 88%, suggesting significant effect of defect density at this range of parameters. Similar trends are observed also for different other initial concentrations for lysozyme and of thaumatin (Figure 5a
b). The essence of all these observations is that within a range of stretching ratio, λ ∼ 1.1
1.4, the coupled effect of wavelength and defect density results in considerable increase in crystal size, d. The insets of Figure 5a
b show that these topographical features affect also the heterogeneity in crystal sizes, which is characterized by estimating the standard deviation of the sizes as measured from typical optical images shown in Figure 3. For example, for thaumatin the standard deviation is calculated to be 32 μm at λ = 1.1 but it decreased to 10 μm at λ = 1.4.

’ SUMMARY It is well-known that defects on surface or perturbations of different types trigger phase change in a supersaturated system. Often such processes result in large number of nucleation of crystals. It is, however, difficult to control this phase change so that nucleation occurs in a restricted manner. In this manuscript, we have generated defects of controlled shapes, size, and number

density on a PDMS substrate which promotes nucleation of crystals. In essence, very large curvature of the defects allows formation of small stable clusters of protein molecules or prenuclii at even a small extent of supersaturation not otherwise possible on a featureless substrate. The process is very similar to capillary condensation on a rainy day: water droplets condense inside a capillary but not on a flat surface (Kelvin effect). In essence we have demonstrated a novel and simple method for crystallizing macromolecules such as proteins over a wide range of sizes ranging from seed crystal to ones amenable to X-ray diffraction studies. The proposed method has several advantages, e.g., in the general area of drug delivery, it will be useful to generate crystals of controlled sizes. On the other hand large crystals will be useful for crystallographic studies via X-ray diffraction. This method requires inexpensive equipment for crystallization, compared to the conventional hanging drop methods. Here low concentration and small volume of protein solution allow for high throughput screening of macromolecular crystallization. Moreover, the crystals can be easily harvested for several engineering and scientific applications.

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected].

’ ACKNOWLEDGMENT This work was supported by the Department of Science and Technology (DST), Government of India. A.S.G. acknowledges DST for the postdoctoral fellowship. We thank the staff of DST Unit on Nanosciences, IIT, Kanpur for their help to carry out AFM scans. ’ REFERENCES (1) Yin, H.
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