Seeding Protein Crystallization with Cross-Linked Protein Crystals

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Seeding protein crystallization with cross-linked protein crystals Er-Kai Yan, Feng-Zhu Zhao, Chen-Yan Zhang, Xue-Zhou Yang, miao shi, Jin He, Ya-Li Liu, Yue Liu, Hai Hou, and Da-Chuan Yin Cryst. Growth Des., Just Accepted Manuscript • DOI: 10.1021/acs.cgd.7b01536 • Publication Date (Web): 08 Jan 2018 Downloaded from http://pubs.acs.org on January 10, 2018

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Crystal Growth & Design

Seeding protein crystallization with cross-linked protein crystals Er-Kai Yana, Feng-Zhu Zhaoa, Chen-Yan Zhanga, Xue-Zhou Yanga, Miao Shia, Jin Hea, Ya-Li Liua, Yue Liua, Hai Houa,b, Da-Chuan Yin*a,b a.

Institute for Special Environmental Biophysics, Key Laboratory for Space Bioscience and

Space Biotechnology, School of Life Sciences, Northwestern Polytechnical University, Xi'an 710072, Shaanxi, People's Republic of China. b

Shenzhen Research Institute of Northwestern Polytechnical University, Shenzhen 518057,

Guangzhou, People's Republic of China.

KEYWORDS: cross-linked protein crystals; protein crystallization; nucleation; screening

The authors declare no conflict of interest. * To whom correspondence may be addressed. Da-Chuan Yin, Email: [email protected], Tel: +86-29-88460254.

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ABSTRACT Protein crystallization is of great importance because protein crystals have a number of different important applications, including large-scale purification of proteins, determination of protein structure, nanoparticle preparation, and theoretical studies of crystallization. An approach often used to efficiently crystallize proteins is the use of nucleants or seeds (small fragments of protein crystals) that can help increase the probability of protein crystallization. Due to the very positive effect that seeding has on protein crystallization, seeds are now widely accepted and utilized in practical protein crystallization. Here, we show that cross-linked protein crystals (CLPCs), which retain the crystal structure but are much more stable than non-cross-linked crystals, can also be used as a new type of seed for promoting protein crystallization. Seeding with CLPCs has effects on both the reproducibility and screening of protein crystals and could improve the optical perfection (well defined facets) of protein crystals and the probability of obtaining protein crystals. In addition, the cross-linked protein crystals may reduce the concentration of protein molecules needed to obtain protein crystals. Furthermore, CLPCs are very stable in air, and no protective medium is necessary for the long-term storage of CLPCs; this feature makes the CLPC seeding method a potentially powerful technique in practical protein crystallization on either a laboratorial or an industrial scale.


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1. INTRODUCTION Protein crystallography remains the most widely used approach to determine the 3-dimensional molecular structures of proteins1-5. The technique routinely used for protein crystallography is X-ray diffraction using a synchrotron radiation facility or an in-house X-ray facility. However, the newly developed XFEL (X-ray free electron laser) and MicroED (electron diffraction using cryoEM) techniques are now attracting more attention due to their unique advantages, such as the ease of time-resolved structure determination at room temperature by XFEL6-8 and fewer requirements for the preparation of protein crystals by MicroED (a single, very small protein crystal is enough for structure determination by MicroED)

9, 10

. ND (neutron

diffraction) is also a very important technique for protein structure determination, as it can provide information about light elements, especially hydrogen, in protein molecules11, 12. All of these techniques require protein crystals as diffraction targets. Obtaining protein crystals is often challenging (and is the so-called bottleneck13, 14 of protein crystallography), so the study of protein crystallization is very important. Protein crystallization also has other very important applications. For example, crystallization of proteins is a desirable process for protein purification and is very useful in industrial production of highly pure, bioactive proteins for use as biopharmaceuticals, especially given the current trend of ABC (Anything But Chromatography)15. Cross-linked protein crystals possess much higher mechanical strength than non-cross-linked ones and show excellent stability in non-aqueous ambient environments; therefore, cross-linked protein crystals are very useful in many applications, such as protein separation16, 17, nanomaterial preparation18, 19, catalysis20, biosensing21, 22. The CLPCs also play an important role in oral delivery of therapeutics23, 24, acting similar to some porous materials, such as proteocubosomes25, pDNA/lipid NP26, the


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self-assembled nanoparticles of cyclodextrins and nonlamellar lipids27, self-assembled multicompartment liquid crystalline lipid carriers28, etc. Protein crystals can also be explored as an excellent confined chemical environment with applications in materials science and bionanotechnology29, 30, such as the production of highly homogeneous nanoparticles18, 19, 31. Furthermore, protein crystallization is a perfect model for studying the mechanism of nucleation and growth of crystals, which is of theoretical importance for studying the crystallization process. Given the large variety of applications in miscellaneous fields and emerging research directions, it is understandable that the development of methods for increasing the chance to obtain protein crystals is still of great importance. The first step to obtaining protein crystals is crystallization screening, which involves the search for suitable chemicals or combinations of chemicals that can help crystallize target proteins. Since the probability of obtaining protein crystals from the screening is usually less than 20%4, it is necessary to develop methods to increase the chance of obtaining protein crystals. Among the many techniques developed to promote crystallization, the use of nucleants or seeds (crystal fragments) has been shown to be a very efficient method. Crystals can nucleate directly from solution; however, it is much easier for crystals to form when some nucleants or seeds are already present in the solution. Nucleants or seeds help proteins crystallize in both the metastable and nucleation zones, as shown in the phase diagram of protein crystallization, while crystallization directly from solution only occurs when the solution is in the nucleation zone, in which the supersaturation is high enough. The metastable zone corresponds to lower saturation, which is preferable for producing higher quality protein crystals32. Therefore, the use of nucleants or seeds is one important way to increase the chances


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of obtaining protein crystals of desirable quality. Many types of nucleants and seeds33-35 have been tested and shown to be useful in protein crystallization. In 1987, McPherson reported a study of the use of nucleants in protein crystallization36. In that study, the effects of 50 different inorganic crystals as nucleants in protein crystallization were examined, and it was found that protein crystallization can be induced by heterogeneous nucleation at much lower supersaturation than needed for spontaneous nucleation. Later, many other nucleants, such as cellulose, dried seaweed, hydroxyapatite37, horse hair powder34, mesoporous bioactive gel-glass (CaO-P2O5-SiO2)38, Langmuir-Schaeffer films39, polymeric films40, porous silicon33, SDB41, and molecularly imprinted polymers (MIPs)42, have been tested and have shown promising effects on the facilitation of protein crystallization37, 43-47, 48

. Similar to nucleants, seeds have also shown impressive effects on the facilitation of protein

crystallization. Seeds are usually small fragments of protein crystals. The history of the utilization of protein crystal seeds goes much further back than that of nucleants. The use of protein crystal seeds can be dated back to 189849 when Hofmeister noted that a crystalline (albumin) product can generally be obtained with rapidity upon addition of some (albumin) crystals to the mixture solution. In 1946, Alderton and Fevold50 showed that lysozyme can be directly crystallized from egg whites using lysozyme seed crystals. In 1971, Davies and Segal51 reported the use of crushed protein crystals as a stock of seeds for subsequent seeding to grow large protein crystals. To date, a variety of different seeding techniques (streak seeding, microseeding/macroseeding, microseed matrix screening, etc.) have been developed and utilized35, 43, 48, 52-65. The source of the seeds can be homogeneous (homogenous seeding uses seeds that are crystals of the target protein) or heterogeneous (heterogenous or cross-seeding uses seeds that are crystals of a protein different from the target protein). All studies have shown that


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seeding is indeed a dramatically powerful tool in protein crystallization, in both screening and optimization processes. As a complementary method, in this paper we propose the use of cross-linked protein crystals (CLPCs) as seeds for facilitating protein crystallization. CLPCs are protein crystals that are chemically cross-linked with a cross-linker (i.e., glutaraldehyde, carbohydrates, thiols, etc.) in their crystalline state66. There are strong covalent bonds formed in the crystals after cross-linking (including intermolecular cross-links and intramolecular cross-links), leading to the consolidation of the crystal structure. Thus, CLPCs possess significantly different properties from the untreated protein crystals and their mechanical stabilities67, 68, thermal stabilities69, 70, and activities in organic solvents71-73 are also significantly enhanced. In addition, CLPCs are insoluble in water and organic solvents71. These excellent properties render CLPCs stable in conventional environments without the need for additional protective measures. Furthermore, CLPCs possess three-dimensionally ordered structures and their diffraction patterns are very similar to those of native crystals. We anticipated that CLPCs may show effects similar to native protein crystal seeds in the induction of protein crystallization. Recently, Koizumi et al74 found that the CLPCs can be used to obtain strain-free protein crystals indicating that CLPCs may be efficiently used as seeds. Since CLPCs have the advantages of being stable and easy to store and manipulate, seeding with CLPCs could be a good tool in protein crystallization. Based on the above considerations, we used CLPCs as seeds to explore their performance in protein crystallization. The effects of CLPC seeds on the probability of protein crystallization were studied, and the mechanism behind the phenomenon is discussed.

2. MATERIALS AND METHODS 2.1 Materials


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Twelve commercial proteins were utilized in this study. Hen egg-white lysozyme was purchased from the Seikagaku Kogyo Company (Japan). Concanavalin A, proteinase K, thaumatin, catalase, α-chymotrypsinogen, α-chymotrypsinogen A II, myoglobin, subtilisin A VIII, pepsin and trypsin were purchased from Sigma-Aldrich (USA). All the purchased proteins were used without further purification. Hemoglobin was purified in our laboratory. Glutaraldehyde and HEPES-Na were purchased from Chemical Reagent Co. Ltd, China. The Index Screening Kit (No. HR2-144) and 96-well microbatch crystallization plates (No.HR3-267) were purchased from Hampton Research (USA). A Gryphon Robot (Art Robbins Instruments, Sunnyvale, CA) was used to conduct the crystallization trials. 2.2. Experimental procedures 2.2.1 Preparation of cross-linked protein crystal seeds (CLPC seeds) We used two methods (preparation via cross-linking of large protein crystals and via direct cross-linking of micro-/nanocrystals) to prepare the CLPC seeds as described below: (1) Preparation of CLPC seeds via cross-linking of large protein crystals (200 to 300 µm in size) Preparation of the CLPC seeds involves the following three steps: (1) Crystallizing the protein to obtain crystals as precursors for the subsequent procedure; (2) cross-linking the crystals to obtain the CLPCs; (3) pulverizing the CLPCs to obtain the CLPC seeds. For the first step, we used the sitting-drop vapor-diffusion method to crystallize the selected proteins (lysozyme, concanavalin A, catalase, proteinase K, and glucose isomerase) for preparing the seeds. The initial crystallization conditions are shown in Table 1. Equal volumes of protein solutions (250 µl) and precipitant solutions (250 µl) were mixed together and then placed in a temperature-controlled chamber at 293 K ± 0.1 K75. The incubation time was 2 days.


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Table 1. Protein crystallization conditions for preparing CLPC seeds via the cross-linking of large crystals. Initial C Protein

T (K)

Buffer

Precipitant

Reference

80 mg ml-1 NaCl

76

(mg ml-1) 0.1 M sodium acetate Lysozyme

70

293 pH 4.60 0.1 M succinic acid

Catalase

12

Concanavalin 12

15%(w/v) PEG 75

293 pH 7.00

3350

0.1 M bis-tris

2%(w/v) PEG 5000

pH 6.50

MME

77

293

A

0.05 M sodium cacodylate, 20%(w/v) PEG Proteinase K

30

293

0.08 M magnesium acetate

76

8000 pH 6.50 0.2 M NaCl, Glucose

0.1 M Tris-HCl 10

isomerase

293

25%(w/v) PEG

78

pH 8.50 3350

In the second step, the obtained crystals were cross-linked using glutaraldehyde. To avoid the generation of flocculent precipitates during cross-linking, the excess protein in the crystallization solution should first be removed. We removed the excess protein in the solution by replacing the crystallization solution with its corresponding precipitant solution. In this process, 500 µl precipitant solutions were added soon after the crystallization solution was removed. This process was repeated twice to ensure that the remaining protein in the solution would not cause obvious flocculation. After removing the excess protein in the solution, 500 µl glutaraldehyde at a volume fraction of 1% was added to the precipitant solution containing the growing protein crystals. The mixture solution was kept in the temperature-controlled chamber at 293 K for 3 h, 6 h, 12 h, and 48 h to obtain different CLPC. These CLPCs were then washed using triple-distilled water. In the third step, the CLPCs were dried in an oven at 323 K for 48 h and then pulverized


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(ground) into fine crystalline particles. The crystalline particles were the seed crystals ready for use in seeding experiments. Before use, these particles were usually dispersed in pure water at 7.5 mg ml-1 to obtain the seed stock solution for seeding experiments. The whole procedure for the preparation of CLPC seeds is shown in Figure S1. (2) Direct preparation of CLPC seeds via cross-linking of protein micro-/nanocrystals This method includes two steps: (1) Preparation of micro-/nanocrystals; (2) Cross-linking of the micro-/nanocrystals. The sitting-drop method was used for the first step. Lysozyme, catalase, and concanavalin A were chosen as the proteins for preparing the seed crystals using this method and their initial experimental conditions are shown in Table 2. The crystallization conditions used for lysozyme were obtained from the literature79. The crystallization experiments were conducted by mixing equal volumes of protein solutions (100 µl) and precipitant solutions (100 µl), and then incubating the mixture at 293 K ± 0.1 K in a temperature-controlled chamber. A large amount of micro-/nanocrystals were obtained within 5-45 min using the crystallization conditions described above. Table 2. Protein crystallization conditions for preparing CLPC seeds via the cross-linking of micro-/nanocrystals. Initial C

T

Protein

Crystallization Buffer

(mg ml-1)

Precipitant

(K)

time (min) 0.9 M sodium chloride, 100 mM acetate buffer pH

Lysozyme

100

293

1% PEG 8000, 0.1 M

5

4.30 sodium acetate pH 3.00 0.1 M succinic acid Catalase

20

293

30%(w/v) PEG 3350

45

4%(w/v) PEG 5000 MME

30

pH 7.00 Concanavalin A

20

293

0.1 M bis-tris pH 6.50


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In the second step, the crystallization solution with protein micro-/nanocrystals was centrifuged (3000 r/min for 3 min) using an Eppendorf 5418 centrifuge (Hamburg, Germany), and the supernatant solution was removed and replaced by its corresponding precipitant solution containing 1% glutaraldehyde. The cross-linking occurred immediately in the solution. After cross-linking, the cross-linked crystals were cleaned using triple-distilled water. Finally, the cross-linked protein crystals were dispersed in water at 7.5 mg ml-1 to obtain the seed stock solution for subsequent use. 2.2.2 Crystallization reproducibility study Lysozyme was used as a model protein to conduct the protein crystallization reproducibility study. The initial concentrations of lysozyme used were 10 mg ml-1, 15 mg ml-1 and 20 mg ml-1. The buffer solution was 0.1 M sodium acetate at pH 4.60 and the precipitant solution was 80 mg ml-1 NaCl. Protein and precipitant solutions were mixed in a 1:1 volume ratio to prepare the crystallization solution. The experimental processes are shown schematically in Figure 1. The sitting-drop vapor-diffusion method was used in this experiment. Before crystallization, the CLPC seeds needed to be applied to the wells of the crystallization plates (Intelli-Plate 96). To achieve this, the stock solution containing the CLPC seeds was dispensed to the wells of the crystallization plate and then the drops (0.5 µl each) were allowed to dry out. After the preparation of the crystallization plates with the seeds, 2 µl of the crystallization solutions from the same mother liquor were then dispensed over the seeds in each well. Sixty microliters of precipitant solution (i.e., 80 mg ml-1 NaCl) was dispensed into each reservoir well. After setting up the crystallization plates with the crystallization solution and the reservoir solution, the plates were immediately sealed using Crystal Clear tape (catalog No. HR4-506; Hampton Research, USA) and placed in the temperature-controlled chamber at 293 K for three days. Images of the


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crystallization droplets were captured using an automated crystal image reader (Xtal-Quest Inc., Beijing, People’s Republic of China).

Figure 1. Schematic illustration of the crystallization experiments. (a) Disperse the CLPC seeds into ultrapure water to obtain a stock solution with seeds at 7.5 mg ml-1; (b) dispense the seed stock solution into crystallization plates; (c) dry out the dispensed droplets in a desiccator to obtain crystallization plates with seeds; (d) set up the crystallization trials; (e) incubate the crystallization plates at a constant temperature in a temperature-controlled chamber; (f) capture the images of the crystallization droplets after incubation.

2.2.3 Crystallization screening study A crystallization screening study was carried out to examine the performance of CLPC seeds in inducing crystallization. Twelve commercial proteins were used to conduct the crystallization screening experiment: lysozyme, proteinase K, concanavalin A, catalase, α-chymotrypsinogen AI, α-chymotrypsinogen, glucose isomerase, thaumatin, pepsin, myoglobin, cellulase, subtilisin A VII. These proteins and their initial concentrations are listed in Table S1. Hemoglobin purified in our laboratory was also used. All proteins were in a buffer solution containing 25 mM HEPES-Na at pH 7.00. The IndexTM (Hampton Research, USA) crystallization screening kit was used. As described above in the section about the crystallization reproducibility study, the crystallization plates with dry CLPC seeds in the wells were prepared


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prior to setting up the crystallization trials. The difference in the study described in this section is that instead of using the CLPCs of a single protein (lysozyme) we used three different seeds or seed mixtures: 1) Seed 1: cross-linked lysozyme crystals; 2) Seed 2: cross-linked crystals of lysozyme and concanavalin A at a 1: 1 volume ratio; 3) Seed 3: cross-linked crystals of lysozyme, concanavalin A and catalase at a 1: 1: 1 volume ratio. These different seeds or seed mixtures were used to prepare crystallization plates with different seeds. The crystallization screening experiments were set up using a Crystal Gryphon (Art Robbins Instruments, Sunnyvale, CA); the crystallization plates were sealed immediately after setting up the trials and were placed in a temperature-controlled chamber at 293 K. After incubation for 3 days, the crystallization plates were taken out of the chamber and images of the crystallization droplets were captured using an automated crystal image reader (Xtal-Quest Inc., Beijing, People ’s Republic of China). To further examine the effects of CLPC seeds on protein crystallization, we prepared a seed mixture of five different CLPCs (CLPCs of lysozyme, concanavalin A, catalase, proteinase K and glucose isomerase at a 1:1:1:1:1 mass ratio; Seed 4) and investigated its effect on protein crystallization. In addition, in order to identify the effect of cross-linking time on the seeding experiments, we used lysozyme crystals that had been cross-linked for 3 h (Seed 5), 6 h (Seed 6), and 12 h (Seed 7). Furthermore, three different directly prepared micro-/nano-CLPC seeds were also used: cross-linked lysozyme micro-/nanocrystals (Seed 8), cross-linked lysozyme and concanavalin A micro-/nanocrystals at a 1:1 mass ratio (Seed 9), and cross-linked lysozyme, concanavalin A and catalase micro-/nanocrystals at a 1:1:1 mass ratio (Seed 10). The 10 seeds are listed in Table 3.


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Table 3. The CLPC seeds used in the study No.

Name

Components of the seeds

Cross-linking time

1

Seed 1

CLPCs of lysozyme

24 h

2

Seed 2

CLPCs of lysozyme and concanavalin A at mass ratio 24 h 1:1:1 CLPCs of lysozyme, concanavalin A and catalase at mass 3

Seed 3

24 h ratio 1:1:1 CLPCs of lysozyme, concanavalin A, catalase, proteinase

4

Seed 4

24 h K and glucose isomerase at mass ratio 1:1:1:1:1

5

Seed 5

CLPCs of lysozyme

3h

6

Seed 6

CLPCs of lysozyme

6h

7

Seed 7

CLPCs of lysozyme

12 h

8

Seed 8

cross-linked lysozyme micro-/nanocrystals

40 min

9

Seed 9

cross-linked lysozyme and concanavalin A 40 min micro-/nanocrystals at mass ratio 1:1 cross-linked lysozyme, concanavalin A and catalase 10

Seed 10

40 min micro-/nanocrystals at mass ratio 1:1:1

3. RESULTS 3.1 Characteristics of the proteins used for preparing the CLPC seeds Five proteins (lysozyme, concanavalin A, catalase, proteinase K, and glucose isomerase) were used to prepare the CLPC seeds. The characteristics of the five proteins are listed in Table S2. It can be seen that the crystals of the five proteins possessed three different space groups. The molecular weights of the five proteins ranged from 14.5 kDa to more than two hundred kDa, and the pI values of the proteins ranged from 4.95 to 11.3. Hence, the proteins used in this study exhibited a wide range of molecular weight and pI values, which may be comparable to a large variety of target proteins.


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3.1.1 Morphologies of the CLPCs Images of the protein crystals were captured under an optical microscope to record the morphologies of protein crystals before and after cross-linking using glutaraldehyde. The fresh lysozyme crystals from the mother liquor were clear and colorless with perfect facet appearance. The cross-linked lysozyme crystals had a pale yellow color that progressively intensified over increasing cross-linking time. 3.1.2 Size distribution of the CLPC seeds DLS is a commonly used method to characterize the size distribution of particles. Here, we used DLS to measure the size distribution of three CLPC seeds (Seeds 1, 2 and 3). The seeds were first dispersed at 7.5 mg ml-1 in saturated solution and then measured using DLS (Nano Zetasizer, Malvern Instruments Ltd, Worcestershire, UK). Figure 2 shows the measurement results. From the figure, it can be seen that approximately 60% of the seeds were in a size range of 800–1300 nm for CLPC seeds of catalase, 1900–2700 nm for CLPC seeds of concanavalin A and 1000–1600 nm for CLPC seeds of lysozyme.

Figure 2. DLS measurements of the size distributions of seeds in saturated sodium chloride solutions. (a) Cross-linked catalase crystal seeds; (b) cross-linked concanavalin A crystal seeds; (c) cross-linked lysozyme crystal seeds.


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3.1.3 Morphologies of the CLPC seeds obtained from the direct preparation of micro-/nanocrystals Figure 3 shows some representative images of the CLPC seeds obtained from the direct preparation of protein micro-/nanocrystals. From the figure, it can be seen that the sizes of the seeds were mostly uniform. The sizes of the CLPC seeds of lysozyme, catalase and concanavalin A were approximately 16 µm, 6 µm, and 2 µm, respectively.

Figure 3. Morphologies of the CLPC seeds obtained from the direct preparation of micro-/nanocrystals. (a) Cross-linked lysozyme crystals; (b) cross-linked catalase crystals; (c) cross-linked concanavalin A crystals.

3.2 Reproducibility study of lysozyme crystallization Due to the poor reproducibility of protein crystallization, crystallization droplets can yield different results even though they come from the same mother liquor80. A number of repeated crystallization experiments (i.e., a reproducibility study) may indicate subtle differences in the crystallization process. In this work, we studied crystallization behavior using 48 identical drops dispensed from the same crystallization solution. Figure 4 shows the morphologies of lysozyme crystals grown at different initial concentrations with and without CLPC seeds. It can be seen that the sizes of the crystals increased with increasing concentration of the lysozyme. More strikingly, the crystals obtained in the presence of CLPC seeds showed large crystal sizes and numbers, and better optical perfection (well defined facets) than the control.


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Figure 4. Morphologies of lysozyme crystals grown at different initial concentrations in the presence or absence of CLPC seeds in the reproducibility study. (a1-c1) Lysozyme crystals grown in the control group; (a2-c2) lysozyme crystals grown in the presence of Seed 1; (a3-c3) lysozyme crystals grown in the presence of Seed 2; (a4-c4) lysozyme crystals grown in the presence of Seed 3. (a1-a4) 10 mg ml-1 lysozyme; (b1-b4) 15 mg ml-1 lysozyme; (c1-c4) 20 mg ml-1 lysozyme.

The crystallization success rate, defined as the ratio of the number of droplets that yield crystals to the total number of droplets81, of lysozyme in the reproducibility study is shown in Figure 5. The figure shows that the crystallization success rate of lysozyme increased with increasing concentration of protein. From Figure 5(a), the crystallization success rate of lysozyme in the presence of CLPC seeds increased significantly compared to the control. In addition, the lower the initial concentration of the protein was, the more significant the difference in the crystallization success rate was.


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Figure 5. Crystallization success rate of lysozyme crystallization with and without CLPC seeds in the reproducibility study. (a) The normalized success rates of lysozyme at different concentrations (10 mg ml-1, 15 mg ml-1, 20 mg ml-1); (b) the averaged normalized success rates.

To verify that CLPCs can indeed promote protein crystallization, we added Seed 1, 2 and 3 to the crystallization solution whose initial protein concentration was 2.5 mg ml-1 (after mixing). We confirmed that the control experiment (without seeds) had no crystals in the droplets incubated for more than 2 weeks. However, we observed readily reproducible crystallization when the seeds were added. The results confirmed that CLPCs can induce crystallization at low concentrations. 3.3 Screening study of protein crystallization 3.3.1 Morphologies of the protein crystals Figure 6 shows the morphologies of several typical protein (lysozyme, proteinase K, glucose isomerase, α-chymotrypsinogen A II, and thaumatin) crystals obtained in the presence and absence of CLPC seeds from the crystallization screening experiments. The crystallization conditions for each protein (either with or without the addition of seeds) were identical for all treatments. It can be seen from Figure 6 that the crystals obtained using the CLPC seeds showed generally larger size and better optical perfection compared to those obtained without the addition of seeds. This result was similar to that seen in the reproducibility study.


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Figure 6. Morphologies of protein crystals grown in the presence and absence of CLPC seeds. (a1-e1) In the absence of seeding; (a2-e2) in the presence of Seed 1; (a3-e3) in the presence of Seed 2; (a4-e4) in the presence of Seed 3. (a1-a4) Lysozyme: 15 mg ml-1, C4 from IndexTM, 35% v/v tacsimate, pH 7.00; (b1-b4) proteinase K: 10 mg ml-1, C9 from IndexTM, 1.1 M sodium malonate, 0.1 M HEPES, 0.5% v/v jeffamine ED-2001, pH 7.00; (c1-c4) glucose isomerase: 7 mg ml-1, E11 from IndexTM, 0.02 M magnesium chloride hexahydrate, 0.1 M HEPES, 22% w/v poly (acrylic acid sodium salt) 5100, pH 7.50; (d1-d4) α-chymotrypsinogen A II: 20 mg ml-1, C4 from IndexTM, 35% v/v tacsimate, pH 7.00; (e1-e4) thaumatin: 20 mg ml-1, H2 from IndexTM, 0.2 M potassium sodium tartrate tetrahydrate, 20%w/v PEG 3350.

When testing other proteins in our experiments, we also observed an improvement in the optical perfection of the crystals. Figure 7 shows examples of morphological improvement of two proteins (pepsin and α-chymotrypsinogen) with and without the addition of seeds (Seed 8, 9 and 10). In the case of pepsin, when there were no seeds added the needle-like crystals appeared to be clustered together (Figure 7(a1)), while in the presence of seeds (Seeds 8, 9, and 10


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corresponding to Figure 7(b1), (c1) and (d1)) rod-shaped single crystals appeared. In the case of α-chymotrypsinogen, the crystals were needle-like in the absence of seeds (Figure 7(a2), while thicker, larger and more faceted crystals appeared (Figure 7(b2), (c2) and (d2)) in the droplets.

Figure 7. Examples of morphology improvement upon the addition of seeds. (a1-a2) In the absence of seeding; (b1-b2) in the presence of Seed 8; (c1-c2) in the presence of Seed 9; (d1-d2) in the presence of Seed 10. (a1-d1) Pepsin: 20 mg ml-1, 0.1 M

sodium acetate trihydrate, 3.0 M sodium chloride, pH 7.50; (a2-d2)

α-chymotrypsinogen: 20 mg ml-1, 0.2 M potassium sodium tartrate tetrahydrate, 20 w/v PEG 3350.

3.3.2 Effect of the addition of CLPC seeds on crystallization screening hits We tested the effect of the addition of CLPC seeds on protein crystallization screening hits. A total of 13 different kinds of proteins (12 commercial proteins and 1 purified in our lab) were used for the study. The crystallization screening kit used was the IndexTM Kit (Hampton Research, USA). Figure 8 shows the crystallization screening hits of the 13 proteins under four conditions: the control (no seeds), and Seeds 1, 2 and 3. Figure 8(a) shows a comparison of the screening hits obtained under the four conditions, and it can be seen that under all the conditions tested, the presence of the seeds resulted in larger numbers of screening hits than the lack of seeds. Myoglobin, in particular, did not yield crystals in the control conditions but did form crystals in the presence of seeds. It can also be seen that the most prominent improvement in the number of crystallization screening hits (60% more than the control) was observed for the


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addition of Seed 3, which was a mixture of three different CLPCs. In addition, we noticed that seeding using the CLPCs of a protein could most significantly improve the screening hits of that same protein. For example, in the crystallization of lysozyme, the number hits increased 31.7% when using CLPCs of the same protein, i.e., lysozyme (Seed 1), while the improvement was 14.63% when using CLPCs containing concanavalin A (i.e., Seed 2), and 2.4% when using CLPCs containing concanavalin A and catalase (i.e., Seed 3).

Figure 8. Comparisons of the number of crystallization screening hits with and without the addition of CLPC seeds. (a) The normalized screening hits; (b) the averaged normalized screening hits.

In our experiments we also noticed that the exact hits were partially complementary when different types of seeds were used. This result indicated that combination of different seeds could help increase the probability of crystallization. Therefore, we performed a verification experiment using a mixture of five different CLPCs (Seed 4; cross-linked crystals of lysozyme, concanavalin A, catalase, proteinase K and glucose isomerase at a 1:1:1:1:1 mass ratio). Figure 9 shows the crystallization screening hits obtained using Seed 4. As seen in the figure, the seed mixture showed significantly higher efficiency (113.16% higher than the control) in facilitating crystallization screening than the CLPCs of any single protein.


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Figure 9. Comparisons of the number of crystallization screening hits with and without the addition of the mixed CLPC seeds. (a) The normalized screening hits; (b) the averaged normalized screening hits. Seed 4 was a mixture of equal masses of CLPCs of five different proteins (lysozyme, concanavalin A, catalase, proteinase K and glucose isomerase).

Cross-linking of protein crystals is a stepwise process; the structures of the crystals may vary upon cross-linking. Excessively cross-linked crystals may lose the native structure of the proteins thus reducing the effectiveness of crystallization, while the incomplete cross-linking of crystals may lead to their dissolution in the crystallization solution, which also decreases the effectiveness of the crystallization. Hence there must exist an optimum cross-linking time for achieving the best crystallization screening results. Based on this hypothesis, we tested the effect of cross-linking time (3 h, 6 h and 12 h cross-linked lysozyme crystals corresponding to Seeds 5, 6, and 7) on crystallization; the results are shown in Figure 10. We can see from the figure that the above hypothesis was true. In this study, the 6 h cross-linking time corresponded to the highest improvement in the number of screening hits.


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Figure 10. Effect of the cross-linking time of the seeds on crystallization screening hits. (a) The normalized screening hits with and without the addition of CLPC seeds; (b) the averaged normalized screening hits with and without the addition of CLPC seeds. Seeds 5, 6 and 7 corresponded to the lysozyme CLPC seeds that were cross-linked for 3 h, 6 h and 12 h, respectively.

Again, since cross-linking is a stepwise process, the reaction always starts from the surface and proceeds to the center of the crystal. Therefore, a cross-linked crystal is always inhomogeneously cross-linked. The CLPC seeds obtained from pulverizing large CLPCs to smaller fragments may exhibit different cross-linking, which may not be a desirable characteristic in ideal seeds. To obtain more homogeneously cross-linked protein crystals the small seed crystals are preferred. Therefore, we prepared protein micro-/nanocrystals and cross-linked them and then used them directly as seeds (Seeds 8, 9 and 10, corresponding to CLPCs of lysozyme; lysozyme and concanavalin A (1:1); and lysozyme, concanavalin A and catalase (1:1:1), respectively. Figure 11 shows the performance of the micro-/nano-CLPCs in facilitating crystallization screening. It can be seen that all of the seeds significantly increased the number of screening hits, and the highest improvement (204.91% higher than the control) was observed when Seed 10 was used. We also noticed that the micro-/nano-CPLC seeds performed better than the pulverized CLPC seeds (Seeds 1-7).


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Figure 11. The effect of micro-/nano-CLPCs, obtained by directly cross-linking micro-/nanocrystals, on crystallization screening hits. (a) The normalized screening hits with and without the addition of CLPC seeds; (b) The averaged normalized screening hits with and without the addition of CLPC seeds. Seeds 8, 9 and 10 corresponded to cross-linked lysozyme micro-/nanocrystals; mixture of cross-linked lysozyme and concanavalin A crystals at mass ratio 1:1; and cross-linked lysozyme, concanavalin A and catalase crystals at a 1:1:1 mass ratio, respectively.

4. DISCUSSION 4.1 The mechanisms of the effects of CLPC seeds on protein crystallization In this work, we have observed the improvement in protein crystallization by adding CLPCs into the crystallization solution. Based on the phenomena observed we propose that the improvement can be attributed to the following main mechanisms: 1) homoepitaxial growth (or direct growth) from the seeds by packing the molecules of the same protein (in the case of homogeneous seeding) onto the crystalline faces; 2) heteroepitaxial growth from the seeds by packing the molecules of different proteins (in the case of cross-seeding or heterogeneous seeding) onto the crystalline faces; 3) nucleation of protein crystals on the porous structure of the CLPCs. 4.1.1 Homoepitaxial growth from CLPC seeds As verified for over one century and now commonly understood, the fragments of protein crystals can be used directly as seeds to promote crystallization of the same protein. We


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hypothesized that cross-linked protein crystals could also act as seeds because they retained the crystal structure, which is crucial for homoepitaxial growth directly from the surface. The lattice constants of CLPCs are very similar to those of protein crystals growing from the mother liquor. According to previous studies, there are minimal or no detectable82 differences in protein structure between CLPCs and native protein crystals. Hence, when the target protein is the same as that of the CLPC seeds crystals grow easily because the protein molecules can be easily incorporated into the crystal structure just as they would be on the surface of a growing crystal of the same protein. The fact that the increase in crystallization screening hits was more prominent using homogeneous seeding compared with using heterogeneous seeding indicates that the process occurs through the mechanism described above. 4.1.2 Heteroepitaxial growth from CLPC seeds The growth of protein crystals from the seeds may also be heteroepitaxial when the target protein is different from the protein of the CLPC seeds. The structures of CLPCs may provide ordered growth sites for the target protein crystal to grow in an orderly way, i.e., the lattices of crystals of the two proteins may match. If the proteins being crystallized are similar to the seed proteins (e.g., mutants) or if the sizes of the molecules and/or the interactions between them are similar to those of the seeds, then the specific capture of protein molecules by binding sites of the seeds may occur, leading to heteroepitaxial growth (homogeneous seeding or cross seeding) and facilitating crystallization. 4.1.3 Nucleation of protein crystals on the porous surfaces of CLPC seeds Heterogeneous nucleants (such as horsehair34, biological porous glass, porous silica33, hydroxyapatite, and SDB microspheres41) have been studied in the field of protein crystallization for many years, and it has been found that heterogeneous nucleants are very helpful in


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facilitating protein crystallization by effectively reducing the nucleation barrier of protein crystallization. Nucleation of protein crystals can occur in the metastable phase, as shown in the phase diagram, so that both the number of crystallization screening hits and the crystal quality can be improved by using heterogeneous nucleants. In the current study, the cross-linked protein crystals can act as heterogeneous nucleants when the target protein is different from the protein forming the CLPCs, and the crystal structure is different from that of the CLPCs. The porous structure of the CLPCs can provide a good surface for the capture of protein molecules (adsorption) so that new nuclei can form on the surfaces of the CLPCs thereby resulting in a higher probability of crystallization. Since the adsorption of protein molecules by the porous structure is a rather common process, the use of porous CLPC seeds is generally helpful in facilitating protein crystallization for several kinds of target proteins. Chayen et al reported38 that mesoporous materials could promote nucleation of proteins. When the pore sizes of materials range from 2.5 to 15 nm, the pores can help the protein nucleate. In the case of the CLPC seeds, the pore sizes range from 0.5 to 10 nm in diameter83, 84, which is similar to a previously reported range (2.5-15 nm)38. Thus, the porous structure of the CLPCs was apparently beneficial for nucleation. 4.2 The benefits of seeding with CLPCs in protein crystallization In this experimental study we observed clear improvements in both crystallization screening hits and crystal morphologies using cross-linked protein crystals as seeds, which show that seeding using CLPCs is potentially useful in protein crystallization both in screening and in optimization. Utilization of CLPC seeds is not only promising in facilitating protein crystallization but is also an advantageous method for the following reasons: 1) High mechanical stability: CLPCs are mechanically stable and possess much higher


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mechanical strength than native protein crystals. This property makes CLPCs strong enough to withstand mechanical contact during handling; so, CLPC seeds can be prepared in advance and be ready for use. This is a very big advantage because no other seeds show such a high stability while still preserving the crystal structure of the protein. 2) High chemical stability: CLPCs are chemically stable. This property makes it possible to transport and store the seeds in the ambient environment for periods of time without any special protective requirements (such as storing them in a special aqueous medium or at a special temperature) for preserving the structure and properties of the seeds. 3) Insolubility in the crystallization solution: the native crystal seeds can only retain a perfect three-dimensional structure in the mother liquor, and they would crack or dissolve in the crystallization solution8585 if their concentration is not sufficiently high. CLPCs, on the other hand, are insoluble in the crystallization solution, so the CLPC seeds do not dissolve when added to the crystallization solution even if the initial concentration of the crystallization solution is low. This property guarantees the applicability of CLPC seeds with any crystallization droplet no matter what the concentration level of the crystallization solution is. 4) Versatile mechanisms for promoting protein crystallization: As discussed above, CLPCs may have effects on seeding, cross-seeding and heterogeneous nucleation; this implies that one type of CLPC seed can be used for the crystallization of many other proteins. Furthermore, the seeds can be mixtures of a number of different CLPCs, which are even more effective at promoting protein crystallization than CLPCs of a single type of protein.


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5. CONCLUSIONS In the current study, we tested the idea of using CPLCs as seeds in protein crystallization. The results confirmed that CLPCs are indeed very effective in facilitating protein crystallization. CPLC seeding can not only promote the crystallization of the same type of protein but also facilitate the crystallization of other types of proteins. The most advantageous feature of using CLPCs as seeds is that they possess much higher mechanical strength and chemical stability than the native protein crystals while retaining the original crystal structure, which guarantees their effectiveness as seeds in protein crystallization. This unique feature allows for the use of CLPCs in a number of different applications: daily protein crystallization screening, optimization in the laboratory or production of highly purified proteins on an industrial scale, which is a common application for crystallization seeds.


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ACKNOWLEDGEMENT This work was supported by National Natural Science Foundation of China (Grant No. U1632126), the Fundamental Research Funds for the Central Universities (Grant Nos.3102016ZY039), China Postdoctoral Science Foundation (Grant No. 2013T60890), the Natural Science Foundation of Shaanxi Province, China (Grant No. 2016JM3012), the Peak Experience Research Programme in Northwestern Polytechnical University (Grant No. 17GH020843), China Postdoctoral Science Foundation (Grant No. 2017M623248), the Fundamental Research Funds for the Central Universities (grant No. 3102017HQZZ028).

AUTHOR INFORMATION Corresponding Author Da-Chuan Yin: Phone: +86-29-88460254. Fax: +81-29-88460254. E-mail: [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. E.K.Y. and D.C.Y. designed research; E.K.Y., F.Z.Z., C.Y.Z., X.Z.Y., M.S., J.H., and Y. L.L. performed research; E.K.Y., Y.L., and H. H. analyzed data; and E.K.Y. and D.C.Y. wrote the paper.

FUNDING SOURCES This work was supported by National Natural Science Foundation of China (Grant No. U1632126) The Fundamental Research Funds for the Central Universities (Grant Nos.3102016ZY039) China Postdoctoral Science Foundation (Grant No. 2013T60890) The Natural Science Foundation of Shaanxi Province, China (Grant No. 2016JM3012) The Peak Experience Research Programme in Northwestern Polytechnical University (Grant No.


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17GH020843) The China Postdoctoral Science Foundation (Grant No. 2017M623248) The Fundamental Research Funds for the Central Universities (grant No. 3102017HQZZ028)

NOTES The authors declare no completing financial interest. SUPPORTING INFORMATION DESCRIPTION Proteins used in the crystallization experiments. Information about the five proteins used as seeds in this study. Schematic procedure for the preparation of CLPC seeds. This material is available free of charge via the Internet at http://pubs.acs.org.


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For Table of Contents Use Only Seeding protein crystallization with cross-linked protein crystals Er-Kai Yana, Feng-Zhu Zhaoa, Chen-Yan Zhanga, Xue-Zhou Yanga, Miao Shia, Jin Hea, Ya-Li Liua, Yue Liua, Hai Houa,b, Da-Chuan Yin*a,b a.

Institute for Special Environmental Biophysics, Key Laboratory for Space Bioscience and Space

Biotechnology, School of Life Sciences, Northwestern Polytechnical University, Xi'an 710072, Shaanxi, People's Republic of China. b

Shenzhen Research Institute of Northwestern Polytechnical University, Shenzhen 518057,

Guangzhou, People's Republic of China.

Cross-linked protein crystals can be useful as nucleants in protein crystallization. Seeding with CLPCs has effects on both the reproducibility and screening of protein crystals and could improve the morphological perfection of protein crystals and the probability of obtaining protein crystals.

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Seeding Protein Crystallization with Cross-Linked Protein Crystals

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