Zwitterion-Immobilized Imprinted Polymers for ... - ACS Publications

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Zwitterion-Immobilized Imprinted Polymers for Promoting the Crystallization of Proteins Yue Xing,†,⊥ Yufeng Hu,‡,⊥ Lun Jiang,§ Zideng Gao,† Zhenhang Chen,§ Zhongzhou Chen,*,§ and Xueqin Ren*,† †

Department of Environmental Sciences & Engineering and §State Key Laboratory of Agrobiotechnology, China Agricultural University, Beijing, P.R. China, 100193 ‡ School of Food and Environment, Dalian University of Technology, Panjin, P.R. China, 124221

Downloaded by NEW YORK UNIV on September 12, 2015 | http://pubs.acs.org Publication Date (Web): August 25, 2015 | doi: 10.1021/acs.cgd.5b00819

S Supporting Information *

ABSTRACT: Zwitterion additives have been used in protein crystallization to prevent the appearance of crystal clusters. Herein, we have developed a novel approach for the immobilization of zwitterion onto molecularly imprinted polymers (MIPs) to yield high-quality single protein crystals. For lysozyme, trypsin, catalase, proteinase K, concanavalin A-type IV, and thaumatin, simply adding the selected zwitterion (3(methacryloylamino)propyl)-dimethyl(3-sulfopropyl) ammonium hydroxide) into the free solution, the crystallization was improved. When further using the zwitterion-immobilized molecularly imprinted polymers (ziMIPs) developed in the current study, the formation of higher quality crystals was facilitated in a shorter time compared with regular MIPs and traditional crystallization trials. Most notably, concanavalin A-type IV, which has nonunique ordered assembly, gave only the form IV structure with higher resolution in the presence of ziMIPs, justifying the superior function of ziMIPs for the ordered assembly of protein molecules. Thus, the ziMIPs could be widely used in protein crystallization.

1. INTRODUCTION The functional activity of an individual protein is determined by its three-dimensional structure and can be modulated, in some cases, by a small molecule or drug.1 The most powerful method currently available for determining the structure of a protein is X-ray crystallography, and this technique is completely reliant on the availability of high quality single crystals.2 The addition of a wide variety of different heterogeneous nucleants, including minerals,3 horse and human hair,4,5 and porous materials6−8 to the first step of the protein crystallization process (i.e., nucleation) has been investigated as a strategy for obtaining high quality single crystals of numerous proteins. However, the inclusion of these materials is seldom helpful for inducing the crystallization of proteins in a reproducible and robust manner because the nucleants generally lack a high level of affinity for the proteins themselves. Molecularly imprinted polymers (MIPs) were recently used as heterogeneous nucleants to facilitate the crystallization of several different proteins.9,10 MIPs can be fabricated from functional monomers and cross-linking agents in the presence of a target template molecule, and consequently exhibit a high level of specificity for the template molecule.11 Based on these results, MIPs have come to be regarded as ideal nucleants for inducing the crystallization of proteins. We previously reported that precipitant-immobilized MIPs could be used to efficiently facilitate the crystallization of proteins.12 With this in mind, it was envisaged that MIPs of this type could be used to integrate © XXXX American Chemical Society

the functionality of MIPs with an immobilized precipitant to better promote the crystallization of proteins. However, progress in this area has been limited because the screening of suitable precipitants is both complicated and timeconsuming. Several reports have been published in the literature to date indicating that the addition of zwitterionic molecules to protein solutions can have a positive effect on the crystallization of the proteins.13,14 Furthermore, it is well-known that zwitterionic additives can be used to facilitate the crystallization of soluble proteins15,16 and have consequently become useful tools for obtaining membrane protein crystals.17 Zwitterionic additives can also be used to prevent the appearance of crystal clusters18 and improve the quality of protein crystals.19 With this in mind, zwitterion-immobilized MIPs could potentially be used to promote protein crystallization. In this study, we have developed an approach to allow for the introduction of zwitterions such as sulfobetaines into MIPs to give the corresponding zwitterion-immobilized molecularly imprinted polymers (ziMIPs). It was hypothesized that ziMIPs bearing active zwitterionic groups on the side chains could be used to combine the functions of MIPs and zwitterions (i.e., not only adsorb protein molecules from the solution leading to Received: June 12, 2015 Revised: August 6, 2015

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DOI: 10.1021/acs.cgd.5b00819 Cryst. Growth Des. XXXX, XXX, XXX−XXX


Crystal Growth & Design

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Figure 1. Photomicrographs of protein crystals obtained with or without MAPDMSAH as additive in reservoir solution: (a) concanavalin A type-IV with MAPDMSAH, (b) concanavalin A type-IV, (c) lysozyme with MAPDMSAH, (d) lysozyme, (e) proteinase K with MAPDMSAH, (f) proteinase K, (g) trypsin with MAPDMSAH, (h) trypsin. Scale bar represents 200 μm. conditions, except the template protein was replaced with 100 μL of deionized water. 2.2.2. Experiments of Protein Crystallization. All of the crystallization experiments were conducted according to the sittingdrop vapor diffusion method in a 96-well plate. All of the protein crystallization trials were performed at 291 K (18 °C). The crystallization trials were performed in five replicates for each set of conditions. The growth of the crystals was observed by optical microscopy (UOP, Chongqing, China). In this study, four series of crystallization trials were conducted to develop a better understanding of the performance characteristics of the ziMIPs. For series 1, which was named CK, the crystallization trials were set up by mixing 1 μL of protein solution with 1 μL of reservoir solution. For series 2, which was conducted as a trial containing a zwitterion solution, 0.2 μL of MAPDMSAH solution was added to a solution containing 1 μL of protein solution and 1 μL of reservoir solution. For series 3, 1 μL of protein solution was initially mixed with 1 μL of reservoir solution, and the resulting mixture was then treated with the different ziMIPs and MIPs. For series 4, 1 μL of protein solution was initially mixed with 1 μL of reservoir solution and 0.2 μL of MAPDMSAH solution, and the resulting mixture was then treated with the different ziMIPs and MIPs. Detailed solution conditions are shown in Table S1 of the Supporting Information. 2.2.3. X-ray Diffraction Data Collection Experiments and Structure Determination. Crystals chosen for analysis by X-ray diffraction were mounted in a Nylon loop and cryoprotected by soaking the crystals in a solution of mother liquor and 30% glycerol. The crystals were then immediately frozen in liquid nitrogen prior to being analyzed. For Concanavalin A-Type IV, native data were collected on beamline BL1A at the Photon Factory and the diffraction data were processed using HKL 2000.20 The structures were solved using the molecular replacement program Balbes21 and were refined using multiple iterations of restrained refinement as well as TLS and the restrained refinement function in Refmac 5.22 The manual rebuilding of the models was carried out using Coot.23 Water molecules were introduced automatically by Coot and the validity of each water molecule was manually determined using the same program. For all of the other proteins, the diffraction data were collected on the beamline 3W1A at the Institute of High Energy Physics, Chinese Academy of Sciences using standard oscillation methods.

highly supersaturated conditions, but also interact with the protein molecule assembled around them to form crystals). Lysozyme, proteinase K, trypsin, catalase, thaumatin, and concanavalin A Type IV were used to evaluate the performance of the ziMIPs in terms of their ability to promote the crystallization of these proteins.

2. EXPERIMENTAL SECTION 2.1. Materials. [3-(Methacryloylamino)propyl]-dimethyl(3-sulfopropyl) ammonium hydroxide, inner salt (MAPDMSAH, 473170), methacrylamide (MAA), N,N′-methylenebis(acrylamide) (MBA), hydroxyethyl methacrylate (HEMA), lysozyme from chicken egg white (62971), proteinase K from Tritirachium album (P6556), trypsin from bovine pancreas (T9201), catalase from bovine liver (C9322), concanavalin A-Type IV from Canavalia ensiformis (Jack bean) (L7647), and thaumatin from Thaumatococcus daniellii (T7638) were all obtained from Sigma-Aldrich and used without further purification. N,N,N′,N′-Tetramethylethylenediamine (TEMED), ammonium persulfate (APS), sodium dodecyl sulfate (SDS), and glacial acetic acid (AcOH) were purchased from Sangon Biotech Co. (Shanghai, China). All the reagents listed above were purchased as the analytical grades. 2.2. Methods. 2.2.1. Synthesis of Zwitterion-Immobilized Molecularly Imprinted Polymers. The ziMIPs were synthesized according to the following procedure: HEMA (39 mg), MAPDMSAH (146 mg), and MBA (6 mg) were dissolved in 300 μL of deionized water, and the resulting mixture was treated with 100 μL of a 12 mg/ mL template protein solution. The prepolymerized solution was then treated with 20 μL of a 10% (w/v) APS solution, and the resulting mixture was purged with nitrogen for 5 min before being treated with 20 μL of a 5% (v/v) TEMED solution. The mixture was then left to polymerize for 18 h at room temperature. After the polymerization process, the gels were ground into crushed gels, which were washed five times with deionized water. The washed crushed gels were then eluted five times with 10% AcOH in SDS solution to allow for the extraction of the template protein. Finally, the crushed gels were washed with deionized water to remove the residual AcOH and SDS. Lysozyme, trypsin, proteinase K, catalase, concanavalin A-Type IV, and thaumatin were used as templates to synthesize MIPs, and the resulting ziMIPs were named L-ziMIP, T-ziMIP, P-ziMIP, C-ziMIP, C4-ziMIP, and Th-ziMIP, respectively. An MIP without a zwitterion group (named L-MIP, T-MIP, P-MIP, C-MIP, C4-MIP, and Th-MIP) was also prepared in the same way, except MAA, HEMA, and MBA were used as functional monomer and cross-linker agents. A nonimprinted polymer (NIP) was also fabricated under the same

3. RESULTS AND DISCUSSION 3.1. Characterization of ziMIPs. To avoid any interference from the template in the crystallization trials, FT-IR was B



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Table 1. Crystallization Results of ziMIPs, MIPs, and Controls with Cognate or Noncognate Proteins at Metastable Conditionsa MAPDMSAH C4-ziMIP L-ziMIP P-ziMIP T-ziMIP Th-ziMIP C-ziMIP C4-MIP L-MIP P-MIP T-MIP Th-MIP C-MIP

Con A IV

lysozyme

proteinase K

trypsin

thaumatin

catalase

0 100 80 0 60 0 20 20 − − − − −

20 60 100 80 80 0 20 − 80 − − − −

0 0 0 80 0 0 0 − − 60 − − −

20 0 20 60 100 20 20 − − − 40 − −

20 40 80 20 20 80 40 − − − − 80 −

0 0 0 0 0 0 60 − − − − − 0

Notes: Successful rates (%) were used to represent the crystallization results. Each experiment was set up with five replicates. − means that no crystallization trials were set up in this condition. Downloaded by NEW YORK UNIV on September 12, 2015 | http://pubs.acs.org Publication Date (Web): August 25, 2015 | doi: 10.1021/acs.cgd.5b00819

a

Figure 2. Photomicrographs of protein crystals obtained with cognate ziMIPs or MIPs: (a) concanavalin A type-IV with C4-ziMIPs, (b) concanavalin A type-IV with C4-MIPs, (c) lysozyme with L-ziMIPs, (d) lysozyme with L-MIPs, (e) proteinase K with P-ziMIPs, (f) proteinase K with P-MIPs, (g) trypsin with T-ziMIPs, (h) trypsin with T-MIPs, (i) thaumatin with Th-ziMIPs, (j) thaumatin with Th-MIPs, (k) catalase with CziMIPs, (l) catalase with C-MIPs. Scale bar represents 200 μm.

about MAPDMSAH. In this study, we have investigated the addition of MAPDMSAH to the reservoir solution to evaluate its preliminary performance on protein crystallization. Concanavalin A-Type IV gave crystals (Figure 1a) with a resolution limit of 1.7 Å within 5 days from the MAPDMSAH solution, whereas no crystals (Figure 1b) were observed during 3 weeks in the control experiment without MAPDMSAH (named CK). Lysozyme crystals with a resolution limit of 2.06 Å (Figure 1c, and Figure S2a in Supporting Information) formed within 48 h in the presence of a 2.7% (w/v) solution of MAPDMSAH in sodium chloride, whereas the corresponding control (CK) afforded no crystals until 2 weeks (Figure 1d). For proteinase

used to monitor the complete removal of the template from ziMIPs. As shown in Supporting Information Figure S1, the spectrum of P-ziMIP was identical to the spectrum of P-ziNIP, and P-MIP had the same spectrum as the P-NIP, demonstrating the complete removal of the template. Besides, the peaks of PziMIP and P-ziNIP at 1200 and 1050 cm−1 were for the R-SO3− vibration of the functional monomer, illustrating the success immobilization of MAPDMSAH onto the ziMIPs. 3.2. Performance of MAPDMSAH in Free Solution Regarding Protein Crystallization. Several zwitterionic molecules have been shown to have a positive impact on the crystallization of proteins,13,14 while there was no research C



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Figure 3. Photomicrographs of protein crystals obtained in the presence of cognate ziMIPs with or without MAPDMSAH solution: (a) lysozyme with MAPDMSAH solution, (b) lysozyme without MAPDMSAH solution. Scale bar represents 200 μm.

C4-MIPs after 48 h (Figure 2b). Crystals of lysozyme formed in the presence of both L-ziMIPs (Figure 2c, and Figure S2b in Supporting Information) and L-MIPs (Figure 2d) with diffraction resolution limits of 1.85 and 2.1 Å, respectively. Proteinase K gave crystals with a resolution limit of 1.1 Å within 10 days when the crystallization experiment was conducted in the presence of P-ziMIPs (Figure 2e, and Figure S 2d in Supporting Information). However, when the crystallization experiment was conducted in the presence of P-MIPs, only twin crystals (Figure 2f) were observed within the same time frame. For trypsin, crystals with a resolution limit of 1.54 Å formed within 48 h when the experiment was conducted in the presence of T-ziMIPs (Figure 2g), but no crystals were observed with T-MIPs (Figure 2h) following an equal culturing period. Thaumatin afforded crystals with diffraction resolution limits of 2.0 and 3.39 Å when the experiment was conducted in the presence of the Th-ziMIPs (Figure 2i, and Figure S2e in Supporting Information) and Th-MIPs (Figure 2j, and Figure S 2f in Supporting Information), respectively. For catalase, crystals with a resolution limit of 3.27 Å formed within 8 days when the experiment was conducted in the presence of CziMIPs (Figure 2k). In contrast, no crystals were formed when the experiment was conducted in the presence of the C-MIPs (Figure 2l), with only minor precipitation being observed. The ziMIPs and the MIPs both exhibited high levels of affinity toward their template proteins. These results therefore indicate that the differences in the crystallization of the proteins could be attributed to the immobilized zwitterions, which could interfere with ionic, hydrogen, or hydrophobic interaction between the proteins. Besides, different phenomena were observed when conventional MIPs and ziMIPs were incorporated respectively (Figure S3 in Supporting Information). For catalase, precipitation appeared quickly in the presence of CMIPs (Figure S3a) and no crystal was obtained. But when using C-ziMIPs, protein solution remained stable and gradually occurred liquid−liquid phase separation (small droplets, Figure S3b), which was the higher rate of supersaturation than precipitation condition. Finally the small droplets transferred into crystals (Figure S3c). What’s more, for trypsin, progression of the formation of phase separation and crystalline aggregation at the protein-rich droplets was also observed (Figure S 3d). All of these results show that the ziMIPs were superior to the traditional MIPs in terms of facilitating the crystallization of the proteins evaluated in the current study. 3.4. Synergistic Effect of MAPDMSAH Solution and ziMIPs in Protein Crystallization. The synergistic effect of a MAPDMSAH solution containing ziMIPs was investigated with the aim of developing a better protein crystallization method. The results revealed that lysozyme gave crystals with a

K, a small single crystal (Figure 1e) was obtained within 24 h using a MAPDMSAH solution, whereas the corresponding control solution yielded no crystals, even after 3 weeks (Figure 1f). The diffraction resolution limit of the proteinase K crystals grown in the presence of MAPDMSAH was found to be 1.3 Å. For trypsin, several crystals (Figure 1g) formed within 3 days in the presence of a MAPDMSAH solution, while twin-crystals (Figure 1h) appeared in the corresponding control (CK) during the same time frame. The diffraction resolution limit of the trypsin crystals grown with MAPDMSAH was 1.7 Å, whereas the twin crystals obtained under the CK conditions cannot be diffracted effectively. Taken together, these results demonstrate that the addition of MAPDMSAH effectively promoted the crystallization of the proteins evaluated in this study, and implied that the immobilization of MAPDMSAH onto MIPs could dramatically enhance the performance of MAPDMSAH and MIPs in terms of their ability to generate crystals. 3.3. Performance of the ziMIPs in Facilitating Protein Crystallization. Several crystallization trials were conducted with the cognate and noncognate ziMIPs to verify the function of the immobilized zwitterion (Table 1). As shown in Table 1, all six of the proteins tested in the current study successfully afforded crystals in the presence of their cognate ziMIPs, while no crystals were observed with the MAPDMSAH solution following the same culture time. This difference in the results could be attributed to the protein molecules being more readily adsorbed onto the ziMIPs than MAPDMSAH. This higher level of adsorption would lead to the formation of enhanced supersaturation conditions, which would favor the interaction of the immobilized zwitterions with the protein molecules to yield crystals. Although concanavalin A-Type IV crystals formed in the presence of C4-ziMIP, L-ziMIP, and T-ziMIP, the highest quality crystals were obtained in the presence of C4ziMIP most likely because of the higher affinity of cognate ziMIPs for the protein. Lysozyme, trypsin, and thaumatin also provided similar results, therefore providing further evidence in support of the advantages of using cognate ziMIPs. Taken together, these results confirm our initial hypothesis that the integration of zwitterions into MIPs could be used to facilitate the crystallization of a wide range of proteins. Crystallization trials were conducted with the ziMIPs and the traditional MIPs to further evaluate the performance characteristics of these two different materials (Table 1). The results revealed that concanavalin A-Type IV produced crystals with a resolution limit of 1.79 Å after 48 h when the crystallization process was conducted in the presence of C4-ziMIPs (Figure 2a). In contrast, no crystals appeared when the experiment was conducted under the same conditions in the presence of the D




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C4-ziMIP are shown in Table 2. It is noteworthy that the resolution limits of the crystals prepared in the current study

resolution limit of 1.28 Å when it was crystallized in the presence of an MAPDMSAH solution containing ziMIPs (Figure 3a, and Figure S2c in Supporting Information), which was higher than the resolution limit achieved using ziMIPs alone (Figure 3b). This result therefore demonstrated that the combination of MAPDMSAH and ziMIPs could, to some extent, enhance the protein quality. However, similar results to this were not observed for the other proteins evaluated in the current study. For example, there were no differences in the qualities of the crystals obtained when proteinase K, trypsin, thaumatin, and concanavalin A-Type IV were crystallized using ziMIPs or a combination of MAPDMSAH and ziMIPs. These results therefore provided further proof of the activity of the immobilized zwitterion. Furthermore, catalase did not afford any crystals when the experiment was conducted in the presence of both MAPDMSAH and ziMIPs, implying that there is no need to add an extra zwitterion when you are using ziMIPs. 3.5. Influence of ziMIPs on Crystal Structure. Concanavalin A-type IV was selected as an ideal candidate to validate the potential of ziMIPs for dealing with challenging, structurally uncharacterized, and highly flexible target proteins. Concanavalin A has been studied extensively and more than 50 X-ray crystal structures of this material have been reported to date.24−27 The results of several previous studies have shown that many structural forms of concanavalin A can appear simultaneously under a range of different crystallization conditions, implying the nonunique ordered assembly of protein molecules. In the current study, the solved structure (Figure 4) of concanavalin A revealed the presence of only

Table 2. Data Collection and Refinement Statistics for Concanavalin A-Type IVa data collection wavelength space group cell dimensions a, b, c (Å) α, β, γ (deg) resolution (Å) Rsym or Rmerge (%) I/σ Completeness (%) redundancy

a

0.9932 C2221

refinement resolution (Å)

101.16, 118.24, 248.67 90.00, 90.00, 90.00 50−1.76 (1.79−1.76) 7.5(55.6)

no. reflections Rwork/Rfree (%)

50−1.76 (1.79−1.76) 132250 20.87/22.38

no. of atoms protein ligand/ion water

7227 8 209

21.32(2.03) 94.6(93.7)

B-factors (Å2) protein

21.258

7.7(6.7)

r.m.s. deviations bond lengths (Å) bonds angles (deg)

0.006 1.167

Values in parentheses are for the highest resolution shell.

were better than those obtained using the polymer-induced heteronucleation method26 where the highest resolution was 2.09 Å.

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CONCLUSIONS We have successfully incorporated zwitterions into MIPs to promote protein crystallization. Based on these results, it is envisaged that the ziMIPs are performing two key roles, including (1) the assembly of the protein molecules to form a highly supersaturated state; and (2) the stabilization of the protein molecules to facilitate the ordered growth of the crystals out of the solution. Furthermore, the results of this study demonstrate that ziMIPs can be used to facilitate the crystallization of proteins to give high quality crystals over a shorter period of time than MIPs or traditional crystallization trials. The effectiveness of this new method was demonstrated by the successful crystallization of the flexible protein concanavalin A, which gave high quality crystals with single structural form. Thus, by immobilizing zwitterions onto MIPs, we have succeeded in developing an effective strategy for the optimization of protein crystals.

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Figure 4. Structure of concanavalin A type IV obtained in this study. The blue spheres represent Ca2+, and the light gray ones represent Mn2+.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.cgd.5b00819. Table and additional figures (PDF)

form IV crystals from the crystals obtained in the presence of the C4-ziMIPs, which highlighted the contribution of the ziMIPs in terms of maintaining the ordered conformation of the protein. The protein structure of concanavalin A determined in this study contains four protein molecules (237 residues), one manganese ion, and one calcium ion in the asymmetric unit. The fold of concanavalin A-type IV in this study adopts the same fold types as 3NWK27 structure using JSmol. Furthermore, the crystals that formed in the presence of noncognate ziMIPs, such as L-ziMIP, were all found to be form IV in structure. The data collection and refinement statistics for the concanavalin A-type IV crystals formed in the presence of

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Tel: +86-10-62734078. *E-mail: [email protected]. Tel: +86-10-62733407. Fax: +86-10-62731016. Author Contributions

Y. Xing and Y. F. Hu contributed equally to this work. Y. F. Hu and X. Q. Ren designed research; Y. F. Hu and Y. Xing E



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(19) Tanaka, S.; Ataka, M.; Kubota, T.; Soga, T.; Homma, K.; Lee, W. C.; Tanokura, M. J. Cryst. Growth 2002, 234, 247−254. (20) Otwinowski, Z.; Minor, W. In Macromolecular Crystallography; Academic Press: New York, 1997. (21) Long, F.; Vagin, A. A.; Young, P.; Murshudov, G. N. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2008, 64, 125−132. (22) Murshudov, G. N.; Skubák, P.; Lebedev, A. A.; Pannu, N. S.; Steiner, R. A.; Nicholls, R. A.; Winn, M. D.; Long, F.; Vagin, A. A. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2011, 67, 355−367. (23) Emsley, P.; Lohkamp, B.; Scott, W. G.; Cowtan, K. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2010, 66, 486−501. (24) Deacon, A. T.; Gleichmann, T.; Kalb (Gilboa), A. J.; Price, H. J.; Raftery, J.; Bradbrook, G.; Yariv, J.; Helliwell, J. R. J. Chem. Soc., Faraday Trans. 1997, 93, 4305−4312. (25) Kantardjieff, K. A.; HoÈchtl, P.; Segelke, B. W.; Tao, F. M.; Ruppb, B. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2002, 58, 735− 743. (26) Kanellopoulos, P. N.; Tucker, P. A.; Pavlou, K.; Agianian, B.; Hamodrakas, S. J. J. Struct. Biol. 1996, 117, 16−23. (27) Foroughi, L. M.; Kang, Y. N.; Matzger, A. J. Cryst. Growth Des. 2011, 11, 1294−1298.

prepared all of MIPs and NIPs; Y. Xing and Z. D. Gao engaged in all the protein crystallization experiments; Y. Xing, Z. H. Chen, L. Jiang, and Z. Z. Chen engaged in the data collection of proteins; Y. Xing, Y. F. Hu, X. Q. Ren and Z. Z. Chen wrote the paper. Notes

The authors declare no competing financial interest. ⊥ X.Y. and H.Y. contributed equally to this work.

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ACKNOWLEDGMENTS This work was supported by the Chinese Universities Scientific Fund (2015SY001), Chinese National Scientific Foundation (21375146, 31370720). We thank the staff at beamline BL1A (Photon Factory) and beamline 3W1A (the Institute of High Energy Physics, Chinese Academy of Sciences) for help with crystallographic data collection.

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ABBREVIATIONS ziMIPs, zwitterion-immobilized molecularly imprinted polymers; MAPDMSAH, [3-(Methacryloylamino)propyl]dimethyl(3-sulfopropyl) ammonium hydroxide, inner salt; MAA, methacrylamide; MBA, N,N′-methylenebis(acrylamide); HEMA, hydroxyethyl methacrylate; TEMED, N,N,N′,N′tetramethylethylenediamine; APS, ammonium persulfate; SDS, sodium dodecyl sulfate

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REFERENCES

(1) Gerdts, C. J.; Tereshko, V.; Yadav, M. K.; Dementieva, I.; Collart, F.; Joachimiak, A.; Stevens, R. C.; Kuhn, P.; Kossiakoff, A.; Ismagilov, R. F. Angew. Chem. 2006, 118, 8336. (2) Chayen, N. E. Curr. Opin. Struct. Biol. 2004, 14, 577−583. (3) McPherson, A.; Shlichta, P. Science 1988, 239, 385−387. (4) D’ Arcy, A.; Mac Sweeney, A.; Haber, A. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2003, 59, 1343−1346. (5) 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. (6) Chayen, N. E.; Saridakis, E.; El-Bahar, R.; Nemirovsky, Y. J. Mol. Biol. 2001, 312, 591−595. (7) Hench, L. L. In Sol-gel silica: Processing, Properties and Technology Transfer; Noyes Publications: London, 1998. (8) Khurshid, S.; Saridakis, E.; Govada, L.; Chayen, N. E. Nat. Protoc. 2014, 9, 1621−1633. (9) Saridakis, E.; Khurshid, S.; Govada, L.; Phan, Q.; Hawkins, D.; Crichlow, G. V.; Lolis, E.; Reddy, S. M.; Chayen, N. E. Proc. Natl. Acad. Sci. U. S. A. 2011, 108, 11081−11086. (10) Reddy, S. M.; Phan, Q. T.; El-Sharif, H.; Govada, L.; Stevenson, D.; Chayen, N. E. Biomacromolecules 2012, 13, 3959−3965. (11) Haupt, K.; Mosbach, K. Chem. Rev. 2000, 100, 2495−2504. (12) Hu, Y. F.; Chen, Z. H.; Fu, Y. J.; He, Q. Z.; Jiang, L.; Zheng, J. G.; Gao, Y. N.; Mei, P. C.; Chen, Z. Z.; Ren, X. Q. Nat. Commun. 2015, 6, 6634. (13) Sauter, C.; Ng, J. D.; Lorber, B.; Keith, G.; Brion, P.; Hosseini, M. W.; Lehn, J. M.; Giege, R. J. Cryst. Growth 1999, 196, 365−376. (14) McPherson, A. In Crystallization of Biological Macromolecules; Cold Spring Harbor Laboratory Press: New York, 1999. (15) Rybin, V.; Zapun, A.; Törrönen, A.; Raina, S.; Missiakas, D.; Creighton, T. E.; Metcalf, P. Acta Crystallogr., Sect. D: Biol. Crystallogr. 1996, 52, 1219−1221. (16) Guan, R. J.; Wang, M.; Liu, X. Q.; Wang, D. C. J. Cryst. Growth 2001, 231, 273−279. (17) Hunte, C.; Michel, H. In Membrane Protein Purification and Crystallization, 2nd ed; Elsevier Academic Press: San Diego, 2003. (18) Hamana, H.; Moriyama, H.; Shinozawa, T.; Tanaka, N. Acta Crystallogr., Sect. D: Biol. Crystallogr. 1999, 55, 345−346. F


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