Crystallization of Proteins at Ultralow Supersaturations Using Novel

Mar 13, 2012 - Surfaces and Particle Engineering Laboratory (SPEL), Department of Chemical Engineering, Imperial College London, South. Kensington ...
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Crystallization of Proteins at Ultralow Supersaturations Using Novel Three-Dimensional Nanotemplates Umang V. Shah, Mark C. Allenby, Daryl R. Williams, and Jerry Y. Y. Heng* Surfaces and Particle Engineering Laboratory (SPEL), Department of Chemical Engineering, Imperial College London, South Kensington Campus, London SW7 2AZ, U.K. S Supporting Information *

ABSTRACT: A series of novel three-dimensional (3D) nanotemplates which have tuned surface mesoporosity and surface chemistry based on the protein of interest have been developed to facilitate protein crystallization. The crystallization of five model proteins systems is reported at hereto the lowest reported protein or precipitant concentrations. These improvements were only possible due to the combined use of optimum pore sizes with appropriate surface chemistries in the preparation of the 3D nanotemplates. The success of this strategy can be ascribed to the specific design of the ordered nanotemplates which are based on known physicochemical properties of the protein and offer an alternate targeted strategy for protein crystallization in contrast to previous methods based on the use of universal nucleants. The use of protein tuned nanotemplates will potentially open up new opportunities for the crystallization and structure determination of high value proteins, as well as opportunities for their separation and purification in downstream bioprocessing.

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unmodified surfaces.8,18 Furthermore, studies on surfaces with ionizable functional groups suggest that functionalized surfaces could promote nucleation and shorten protein crystallization times while also optimizing protein consumption levels.7,9,19,20 These studies have highlighted the contribution of surface properties in heterogeneous nucleation, though there is little mechanistic understanding of how this impacts on protein crystallization. However, the effects of nucleant surface chemistry or surface porosity on supersaturation required to obtain protein crystals have been considered in isolation. To the best of our knowledge, no previous studies have combined the effect of specific surface porosity at nanometer length-scale and surface chemistry on crystallization of proteins at lower supersaturation. This study proposed to examine the combined effect of surface porosity and surface chemistry on crystallization of proteins at the protein concentration range usually obtained from the cell lines and bioreactors.21,22 In the present work, surfaces with controlled pore diameter, narrow pore size distribution, and surface chemistry (functional end groups), which are referred as 3D nanotemplates, have been prepared and deployed to study the crystallization of five extensively reported model protein systems: lysozyme, thaumatin, human serum albumin (HSA), concanavalin A, and catalase.

or many decades, efforts in understanding the crystallization of biological macromolecules have been focused toward obtaining good diffraction quality single crystals for protein structural determinations. Structures are key in determining their biological functions and ultimately potential opportunities for therapeutic interventions.1,2 However, there are currently no comprehensive theories, models, or even universal empirical data sets, which can support the systematic design of the necessary crystallization screens for biological macromolecules.3 Instead, current approaches rely on a diverse set of highly empirical principles.2 Empirically based crystallization screening approaches by their very nature require a relatively substantial amount of protein samples, while providing no guarantees of success. It is difficult and cost intensive to prepare large amounts of complex proteins, and hence screening for suitable crystallization conditions and obtaining diffraction quality crystals is still the bottleneck in protein structure determination.4,5 Heterogeneous nucleants are well-known to reduce the change in free energy required to induce nucleation and hence obtain crystals at low solute concentration. Different surface based approaches have been reported for obtaining crystals of diffraction quality for structure determination with the effects of nucleant surface porosity, surface roughness, or surface chemistry being studied.6−11 For example, disordered porous surfaces having wide pore size distribution have been reported to promote nucleation.12−15 Biopolymers and biominerals are also reported to induce protein crystallization at relatively low protein concentrations.16,17 Surfaces with charged functional groups have been used to induce protein nucleation at lower protein concentration than the same required for untreated/ © 2012 American Chemical Society

Received: September 11, 2011 Revised: February 16, 2012 Published: March 13, 2012 1772

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has proven to be difficult.13 HSA has not been previously crystallized using porous heterogeneous nucleants. All proteins and other chemicals were purchased from Sigma Aldrich and used without further purifications. Crystallization trials were carried out using the hanging drop vapor diffusion method in which a droplet consisting of equal volumes of protein and precipitant solution were deposited on the surface of 3D nanotemplates while at the same being equilibrated with the precipitant solution. The crystallization conditions for the protein system under investigation are listed in Table 2. The protein concentration in the droplet was reduced step by step until no crystals were observed on the template surfaces over a period of two weeks. Detailed information about the proteins and chemicals used and method employed for crystallization can be obtained in the Supporting Information. Table 3 reports a comparison of experimental results obtained in this study with other studies reporting either effects of surface chemistry or surface porosity of nucleant surfaces, where well-faceted crystals were obtained avoiding uncontrolled mass crystallization. Details of the results obtained with different model protein systems under investigation are summarized as follows.

For the preparation of these 3D nanotemplates, a sacrificial template synthesis based around previously reported methods was employed.23 In order to tune the substrate porosity and to obtain the necessary ordered mesostructure, triblock copolymer surfactants were used as sacrificial templates. The surface chemistry of the resultant mesoporous glasses were subsequently modified using different organo-silanes. Preparation details are described in Supporting Information. The surface chemistry of the mesoporous surfaces prepared were modified using the method reported by Feng et al.26 Supporting Information describes a detailed method of surface functionalization. Water contact angle data on the modified surfaces are reported in Table 1. The hydrophobicity of the Table 1. Contact Angle of Water on Functionalized Mesoporous Templates (pore diameter 4−6 nm) 3D nanotemplate functionalized with

contact angle

hydroxyl group amino group chloro group phenyl group dodecyl group

11.3° ± 2.8° 33.2° ± 4.1° 82.6° ± 3.5° 109.1° ± 4.9° 128.6° ± 3.2°



CONCANAVALIN A Concanavalin A is a tetramer, which has a molecular weight of 106 kDa, was initially crystallized on surfaces with a pore diameter of 10−12 nm at a protein concentration of 17.5 mg/ mL. At 2.0 mg/mL concentration, crystals were only observed on a functionalized 3D nanotemplate with a specific pore diameter of 10−12 nm. No crystals were observed over the same period of time on any other 3D nanotemplates as well as the control nonporous glass surface. It is worth noting that at protein concentrations of 5.0 mg/mL, well-faceted individual diffraction quality crystals were observed on surfaces with a 10−12 nm pore diameter, whereas uncontrolled crystallization was observed on other nanoporous surfaces. Further reductions in the protein concentration to 1.5 mg/mL resulted in crystals only forming on the surfaces with a pore diameter of 10−12 nm and functionalized with dodecyl functional group. Surfaces with the same pore diameter and other surface chemistry failed to induce crystallization at this concentration. Indeed at 1.5 mg/ mL no crystals were observed within 15 days on surfaces other than those having a pore diameter of 10−12 nm with dodecyl function end groups. The results discussed here were repeated at least nine times.

surface found to increase in hydroxyl < amino < chloro < phenyl < dodecyl order for different functional groups, which is consistent with the order reported in different literature reports.27,28 The templates were then characterized using N2 sorption BET/BJH methods and transmission electron microscopy (TEM).24,25 Figure 1 shows the pore size distribution and



THAUMATIN Thaumatin is a low calorie sweetener having a molecular weight of 22 kDa. Thaumatin crystals were initially obtained on the templates with a pore diameter of 3−4 nm at a protein concentration of 11.0 mg/mL. Reducing protein concentration down to 4.0 mg/mL resulted in crystal formation on functionalized surfaces with 3−4 nm pore diameters, although the crystal density and sizes varied for different surfaces. Further reductions in protein concentration to 2.0 mg/mL resulted in crystals only forming on untreated 3D nanotemplates having 3−4 nm pore diameters which had hydroxyl functional end groups. These crystals have formed at concentrations on par with the lowest protein concentration reported in the literature, although the literature reported crystallization was with a 3 times higher precipitant

Figure 1. Pore size distribution of 3D nanotemplate surfaces prepared.

Figure 2 shows TEM images of 3D nanotemplates used, confirming both the narrow pore size distribution and the wellordered mesostructures obtained by the sacrificial template methods deployed. Lysozyme, thaumatin, HSA, concanavalin A, and catalase are model proteins that have been extensively studied in crystallization for structure determination and method development related studies over the past six decades. These extensively studied protein systems were selected to understand combined effect of nucleant surface porosity and surface functional end groups. The crystallization of concanavalin A using porous surfaces with wide pore size distribution, which are considered as suitable candidates for universal nucleants, 1773

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Figure 2. TEM Images of 3D nanotemplates prepared: (a) Type-II, (b) Type-III (scale bar 20 nm).

other heteronuclei in the literature.29 The experiments were repeated at least five times.

Table 2. Crystallization Conditions under Which Protein Crystals Obtained on Surfaces of 3D Nanotemplates protein lysozyme

molecular weight (kDa) 14.7

thaumatin human serum albumin

22 67

concanavalin A

106

catalase

232

buffer solution (solvent water) 0.1 M sodium acetate pH 4.5 0.05 M PIPES pH 6.8 0.05 M monopotassium phosphate pH 5.15 0.01 M Tris pH 8.5; 0.02 M CaCl2; 0.02 M MnCl2 0.1 M tris pH 8.4



LYSOZYME Lysozyme, a globular protein having a molecular weight of 14.7 kDa, has been extensively studied, including many fundamental studies related to macromolecular crystallization. Crystallization of lysozyme is obtained on the templates with 3−4 nm pore diameters with both hydroxyl as well as dodecyl functional groups at a protein concentration as low as 2.5 mg/mL. Though lysozyme has been extensively studied, this is the lowest concentration reported for heterogeneous crystallization in the literature.9 The experiments were repeated at least nine times. The time elapsed for optical observation of first crystals on the template surfaces was observed to increase with decreasing protein concentration, as reported elsewhere in the literature. An analysis of the relative induction time is detailed in Table 4. Results referred to above were undertaken in a minimum as triplicates, so as to reduce the risk of spurious crystallization results due to contaminants or the presence of other adventitious materials. All of the crystals obtained on the surfaces of 3D nanotemplates at these very low protein concentrations reported here were observed to be individual well-faceted diffraction quality crystals. Figure 3 show reflective light micrographs of crystals obtained at the lowest concentration on surfaces having specific pore diameter and functional end groups. No aggregation or uncontrolled mass crystallization was observed. Surface selective crystallization of protein molecules is evident from the results reported here. Proteins of lower molecular weight and positive surface charge, that is, lysozyme (14.7 kDa) and thaumatin (22 kDa), crystallized at the hereto lowest reported concentration on surfaces with pore diameters in the range of 3−4 nm and hydroxyl functional groups. Human serum albumin of molecular weight 67 kDa was observed be crystallized at the lowest concentration on surfaces with pore diameters of 4−6 nm and hydroxyl functional groups. For proteins of a higher molecular weight, concanavalin A (106 kDa) and catalase (232 kDa), crystals were obtained on surfaces of pore diameter in the range of 10−12 nm. Details of

precipitant solution (solvent buffer) 1.1 M NaCl 0.34 M Na-K tartrate 5% (w/v) PEG 4K

1 M (NH4)2SO4 in 0.02 M Tris pH 8.0 5% (w/v) PEG 4K; 5% (v/ v) 2-methyl-1,3 propanediol (MPD)

concentration than those reported in the current study.7 The results discussed here were repeated at least four times.



CATALASE



HUMAN SERUM ALBUMIN

Templates with a 10−12 nm pore diameter and a range of different surface chemistries resulted in diffraction quality protein crystals of catalase, which is a tetrameter with a molecular weight of 232 kDa, at an initial protein concentration of 6.0 mg/mL. The crystals size, density, and habit vary for different surface-modified templates. A further reduction in protein concentration to 5.0 mg/mL resulted in crystal formation only on surfaces with 10−12 nm pore diameters and amino functional end groups. At least three repeats were carried out.

Human serum albumin (HSA), which has a molecular weight of 67 kDa, was crystallized at an initial protein concentration of 100.0 mg/mL. The crystals were observed on templates with a 4−6 nm pore diameter and hydroxyl surface groups. Following a step-by-step reduction in protein concentration, the lowest protein concentration at which the crystals were obtained is 20.0 mg/mL on surface with a 4−6 nm pore diameter and hydroxyl surface groups. This result is an order of magnitude lower than the best reported values for HSA crystallization on 1774

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no comparable data availabled 11.513 10−12 nm; −NH2 5.0 catalase

porous silicon (5−10 nm)

no data available 10−12 nm; −CH3 1.5

Table 4. Comparison of Results Obtained in This Study with Induction Times Reported in the Literature for Crystallization of Proteins under Influence of Either Nucleant Surface Porosity or Surface Chemistry at Lowest Reported Protein Concentration under Similar Crystallization Conditions

a Data in column 4 and column 6 are the lowest concentration of proteins reported in the literature, at which the crystals appeared on the relevant nucleant surfaces. bComparative analysis of the protein source used in the present study and the same used in the study referred to in column 4 and column 6 is provided in the Supporting Information Table S2. cCrystallization was carried out at higher precipitant concentration as compared to the same used in this study. For thaumatin the precipitant concentration used is 3 times higher (1 M sodium−potassium tartrate), whereas for concanavalin A the same is 1.5 times higher (1.5 M ammonium sulfate). dNo comparable data are available under similar crystallization conditions. Data are only available in varying crystallization conditions with different buffers and precipitants.

54%

70%

mica surfaces modified with -NH2 functional end group/sulfonated polystyrene no comparable data availabled 5.09c

255.029 no data available 4−6 nm; −OH 20.0

human serum albumin concanavalin A

no data available

92%

equalc 2.07c 16.013 3−4 nm; −OH 2.0 thaumatin

15.013

porous silicon (5−10 nm) porous silicon (5−10 nm) no data available

5.09

mica surfaces modified with -NH2 functional end group mica surfaces modified with -NH2 functional end group silicate

50%

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3−4 nm; −OH 2.5

protein

lysozyme

relative low protein concentration required for crystallization type of surfaces and surface functional end groups used protein concentration (nucleants with functionalized surface chemistry) (mg/mL) type of disorderedporous surfaces used protein concentration (nucleants with disordered surface porosity) (mg/mL) pore diameter and surface functional end groups protein concentration (current study) (mg/mL)

Table 3. Comparison of Results Obtained in This Study with Lowest Protein Concentration Reported in the Literature for Obtaining Protein Crystals As a Result of Either Nucleant Surface Porosity or Surface Chemistry under Similar Crystallization Conditionsa,b

Crystal Growth & Design

protein lysozyme thaumatin human serum albumin concanavalin A catalase

time elapsed for observation of first crystals on 3D nanotemplates (h)

induction time reported in the literature (lowest protein concentration reported for system using either nucleants with disordered surface porosity or surface chemistry)a

72−84 24−30 36−48

48 h9 18 h7 no data available

24−36 72−84

22 h9 no data available

a

Induction time reported in the studies reporting crystallization of proteins at lowest protein concentration, corresponding to Table 3; column 6 for lysozyme, thaumatin, HSA, concanavalin A, and Table 3; column 4 for catalase.

protein radius of gyration and surface charge as well as 3D nanotemplate pore diameter and surface charge under crystallization conditions, which resulted in crystals at the lowest protein concentration, is described in Table 5. According to the Gibbs−Thompson expressions, heterogeneous nucleation requires a smaller change in the free energy to overcome the free energy barrier than required for the same formation of homonuclei.30 Hence, in the presence of a heteronuclei, the protein crystals can be obtained at lower critical supersaturation levels. Recently, Tosi et al. experimentally demonstrated that heterogeneous nucleation is not critically effective at higher levels of protein concentration/ supersaturation.31 On the basis of experimental results obtained here, we confirm the previous stated view that the homogeneous nucleation mechanism may dominate at higher degrees of supersaturation, whereas at lower degrees of supersaturation heterogeneous nucleation is prevalent. Different mechanisms have been proposed for the role of either surface porosity or surface chemistry on nuclei formation, although none of these mechanisms takes into account the combined effect of surface porosity and surface chemistry described here.7,11,14 The crystallization of proteins reported here at these low supersaturation can be attributed to the combined influence of surface chemistry and surface porosity, which can more logically be explained using recently proposed two-step nucleation mechanism.32 According to the two step mechanism, transformation from completely disordered liquid phase to the highly ordered solid phase is proposed via a high dense amorphous liquid phase. Vekilov et al. argued that the logically more energetically favorable pathway to phase transition follows two ordered parameters, namely, concentration and structure fluctuation. The sequential pathway corresponds to the formation of a dense liquid phase, which has significantly higher concentration of protein molecules as compared to the bulk liquid. In order to nucleate the stable high dense liquid phase, it is essential to cross the free energy barrier. Once this stable high dense liquid phase is formed, the crystals nucleate following structural transformation.33,34 Intermolecular interactions between the solvated proteins and the surfaces of the mesopores 3D nanotemplates are a 1775

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Figure 3. Crystals of different proteins obtained on surfaces with controlled pore diameter and surface functional end groups. (a) Thaumatin crystals on surface with a pore diameter of 3−4 nm and −OH functional end group. (b) Human serum albumin crystals on surface with a pore diameter of 4−6 nm and −OH functional end group. (c) Concanavalin A on surfaces with a pore diameter of 10−12 nm and −CH3 functional end group, and (d) catalase on surfaces with a pore diameter of 10−12 nm and −NH2 functional end group (scale bar 200 μm).

Table 5. Relation between Surface Porosity, Surface Charge of 3D Nanotemplates, which Resulted in Crystallization at Lowest Concentration, Protein Radius of Gyration, and Surface Charge protein lysozyme thaumatin human serum albumin concanavalin A catalase

3D nanotemplate surface porosity (range of pore diameter) and functional end groups

3D nanotemplate − surface charge

2× radius of gyration (nm)

protein charge in crystallization condition

3.239 3.540 5.441

+ve7 +ve7 neutral or very weak −ve42

3 nm ≤ d ≤ 4 nm; −OH 3 nm ≤ d ≤ 4 nm; −OH 4 nm ≤ d ≤ 6 nm; −OH

−ve −ve −ve

6.343 8.144

neutral or very weak +ve7 −ve45

10 nm ≤ d ≤ 12 nm; −CH3 10 nm ≤ d ≤ 12 nm; −NH2

very weak −ve +ve

function of relative surface charges of the template surface and the proteins at the crystallization pH employed. As per the data shown in Table 5, surface properties resulting in attractive interaction between protein and surface of 3D nanotemplates directly influence, and in our case lowers, the surface free energy barrier necessary for the formation of intermediate amorphous high dense phase from completely disordered liquid phase. This high dense liquid phase, which has a substantially higher protein concentration, is expected to be formed in the proximity of the surface of the template.35−37 Once the high density liquid phase is formed, the next step is the formation of an ordered solid phase from it.32,35 Considering the critical nuclei size as an inverse function of the degree of supersaturation, at such high protein concentrations the critical nucleus size can be minimum, although it becomes extremely important to control the process of nucleus formation.30 Specifically tuned porosity of the 3D nanotemplate deployed here plays an important role in stabilizing the nuclei within the mesopores by local immobilization. If pores available are comparable to the dimensions of the specific protein of interest, then the protein diffuses in the mesopores and local

immobilization promotes the formation of nuclei by substantially lowering the free energy barrier.38 Once the nucleus is formed, functional groups on the surface may stabilize the already formed nuclei by interacting with a specific crystal face.35 It is assumed that the distribution of the functional groups on the surface is not uniform and that this random distribution of functional groups offers many different potential patterns of interaction with the crystal nuclei. Table 5 summarizes details of radius of gyration and surface charges of the protein systems under investigation as well as pore diameters and surface charges of the 3D nanotemplates employed, which agrees with the mechanism proposed herewith. The crystallization of five model proteins systems is reported at hereto the lowest reported protein or precipitant concentrations by employing novel 3D nanotemplates. Individual diffraction quality crystals of concanavalin A were obtained at three times less than previous concentrations reported, whereas crystals of human serum albumin were obtained at an order of magnitude lower protein concentration at the same conditions. In summary, it has been demonstrated 1776

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(12) Asanithi, P.; Saridakis, E.; Govada, L.; Jurewicz, I.; Brunner, E. W.; Ponnusamy, R.; Cleaver, J. A. S.; Dalton, A. B.; Chayen, N. E.; Sear, R. P. ACS Appl. Mater. Interfaces 2009, 1, 1203−1210. (13) Chayen, N. E.; Saridakis, E.; El-Bahar, R.; Nemirovsky, Y. J. Mol. Biol. 2001, 312, 591−595. (14) Chayen, N. E.; Saridakis, E.; Sear, R. P. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 597−601. (15) Rong, L.; Komatsu, H.; Yoshizaki, I.; Kadowaki, A.; Yoda, S. J. Synchrotron Radiat. 2004, 11, 27−29. (16) Georgieva, D. G.; Kuil, M. E.; Oosterkamp, T. H.; Zandbergen, H. W.; Abrahams, J. P. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2007, 63, 564−570. (17) Thakur, A. S.; Robin, G.; Gregor, G.; Saunders, N. F. W.; Newman, J.; Martin, J. L.; Kobe, B. PLoS ONE 2007, e1091. (18) Tsekova, D.; Dimitrova, S.; Nanev, C. N. J. Cryst. Growth 1999, 196, 226−233. (19) Tang, L.; Huang, Y. B.; Liu, D. Q.; Li, J. L.; Mao, K.; Liu, L.; Cheng, Z. J.; Gong, W. M.; Hu, J.; He, J. H. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2005, 61, 53−59. (20) Pham, T.; Lai, D.; Ji, D.; Tuntiwechapikul, W.; Friedman, J. M.; Randall Lee, T. Colloids Surf., B 2004, 34, 191−196. (21) Wurm, F. M. Nat. Biotechnol. 2004, 22, 1393−1398. (22) Hacker, D. L.; De Jesus, M.; Wurm, F. M. Biotechnol. Adv. 2009, 27, 1023−1027. (23) Zhao, D.; Feng, J.; Huo, Q.; Melosh, N.; Fredrickson, G. H.; Chmelka, B. F.; Stucky, G. D. Science 1998, 279, 548−552. (24) Brunauer, S.; Emmett, P. H.; Teller, E. J. Am. Chem. Soc. 1938, 60, 309−319. (25) Barrett, E. P.; Joyner, L. G.; Halenda, P. P. J. Am. Chem. Soc. 1951, 73, 373−380. (26) Feng, X.; Fryxell, G. E.; Wang, L.-Q.; Kim, A. Y.; Liu, J.; Kemner, K. M. Science 1997, 276, 923−926. (27) Delmas, T.; Roberts, M. M.; Heng, J. Y. Y. J. Adhes. Sci. Technol. 2011, 25, 357−366. (28) Delmas, T., MSc Thesis, Imperial College London, 2008. (29) Takehara, M.; Ino, K.; Takakusagi, Y.; Oshikane, H.; Nureki, O.; Ebina, T.; Mizukami, F.; Sakaguchi, K. Anal. Biochem. 2008, 373, 322− 329. (30) Gibbs, J. W. Collected Works, Thermodynamics; Yale University Press: New Haven, 1948; Vol. 1. (31) Tosi, G.; Fermani, S.; Falini, G.; Gavira, J. A.; Garcia Ruiz, J. M. Cryst. Growth Des. 2011, 11, 1542−1548. (32) Vekilov, P. G. Nanoscale 2010, 2, 2346−2357. (33) Vekilov, P. G. J. Cryst. Growth 2005, 275, 65−76. (34) Wolde, P. R. t.; Frenkel, D. Science 1997, 277, 1975−1978. (35) Dey, A.; Bomans, P. H. H.; Müller, F. A.; Will, J.; Frederik, P. M.; de With, G.; Sommerdijk, N. A. J. M. Nat. Mater. 2010, 9, 1010− 1014. (36) Gebauer, D.; Cölfen, H. Nano Today 2011, 6, 564−584. (37) Gebauer, D.; Völkel, A.; Cölfen, H. Science 2008, 322, 1819− 1822. (38) Shah, U. V.; Williams, D. R.; Heng, J. Y. Y. Cryst. Growth Des. 2012, DOI: 10.1021/cg201443s. (39) da Silva, M. A.; Itri, R.; Arêas, E. P. G. Biophys. Chem. 2002, 99, 169−179. (40) Kuznetsov, Y. G.; Konnert, J.; Malkin, A. J.; McPherson, A. Surf. Sci. 1999, 440, 69−80. (41) Olivieril, J. R.; Craievich, A. F. Eur. Biophys. J. 1995, 24, 77−84. (42) Nimai, C. N.; Kwanwoo, S. Nanotechnology 2008, 19, 265603. (43) Pallarola, D.; Queralto, N.; Knoll, W.; Ceolín, M.; Azzaroni, O.; Battaglini, F. Langmuir 2010, 26, 13684−13696. (44) Malmon, A. G. Biochim. Biophys. Acta 1957, 26, 233−240. (45) Lee, B.; Kim, S.; Cho, J. Macromol. Res. 2011, 19, 635−638.

that novel 3D nanotemplates, which have tuned specific surface porosity and surface chemistry, have the ability to significantly lower the critical protein concentrations required to induce controlled heteronucleation and crystallization. The approach presented here can be used to engineer surfaces based on the understanding of protein molecule dimensions and the specific interactions between different surface functional groups and target protein molecule for the crystallization of protein at the lowest degrees of supersaturation.



ASSOCIATED CONTENT

S Supporting Information *

Description of preparation of 3D nanotemplate surfaces, functionalization of surfaces, and crystallization of proteins. Details of the reagents used for preparation of functionalized monolayer on nanoporous glasses (Table S1); comparative analysis of the protein source used in the present study (Table S2); comparative analysis of lowest protein concentration reported in current study with the same reported as lowest protein concentration in the past (Figure S1). This material is available free of charge via the Internet at http://pubs.acs.org.



AUTHOR INFORMATION

Corresponding Author

*Tel: +44 (0)20 7594 0784. Fax: +44 (0)20 7594 5700. E-mail: [email protected]. Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS The authors acknowledge Miss Shanshan Huang for experimental work related to lysozyme, Dr. Jeremy Moore for the single crystal X-ray diffraction analysis, Prof. Jayne Lawrence and Dr. Laila Kudsiova, Kings College London, for fruitful discussions and zeta potential measurements of templates prepared in this study. U.V.S. acknowledges the Education Department, Government of Gujarat, Gandhinagar, India, for granting a secondment to undertake these studies. The support of BBSRC and the Bioprocessing Research Industry Club (BRIC) (BB/F004990/1) for this research work is also gratefully acknowledged.



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