Utilization of Cyclodextrins and Its Derivative Particles as Nucleants for

Subscriber access provided by UNIVERSITY OF ADELAIDE LIBRARIES

Article

Utilization of cyclodextrins and its derivative particles as nucleants for protein crystallization Xue-Zhou Yang, Chen-Yan Zhang, Qianjin Wang, Yun-Zhu Guo, Chen Dong, Er-Kai Yan, Wen-Jing Liu, Xi-Wang Zheng, and Da-Chuan Yin Cryst. Growth Des., Just Accepted Manuscript • DOI: 10.1021/acs.cgd.7b00455 • Publication Date (Web): 20 Oct 2017 Downloaded from http://pubs.acs.org on October 23, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Crystal Growth & Design is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Crystal Growth & Design

Cover page The Title: Utilization of cyclodextrins and its derivative particles as nucleants for protein crystallization The Author: Xue-Zhou Yanga, Chen-Yan Zhanga,*, Qian-Jin Wangb, Yun-Zhu Guoa, Chen Donga, Er-Kai Yana, Wen-Jing Liua, Xi-Wang Zhenga, and Da-Chuan Yina,* a

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

School of Life Sciences, Northwestern Polytechnical University, Xi’an 710072, Shaanxi, PR China b

Shaanxi Energy Institute, Xianyang 712000, Shaanxi, PR China

Cyclodextrins can be useful as nucleants due to the ease of modifying them to suit the crystallization of different proteins, and they can be explored for use in the mass purification of proteins for the biopharmaceutical industry. We conducted different kinds of cyclodextrins on protein crystallization. It was verified that protein crystallization is indeed promoted by adding cyclodextrins. The results also indicated that cyclodextrins exhibited the best performance when the protein in solution had the opposite charge to that of the cyclodextrins. Furthermore, this phenomenon pointed toward a way to find new nucleants based on the overall charge of proteins in a solution: when the zeta potential of cyclodextrin particle was opposite to overall charges of the targeted protein, the nucleants was facilitated. Da-Chuan Yin School of Life Sciences, Northwestern Polytechnical University 127 Youyixi Rd, Xi'an, Shaanxi, PR China Tel.: 86-29-88460254; Fax.: 86-29-88460254 Email: [email protected] http://www.bmlnwpu.org/us/member/yindachuan-e.html

ACS Paragon Plus Environment

1


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 60

Title page The Title: Utilization of cyclodextrins and its derivative particles as nucleants for protein crystallization The Author: Xue-Zhou Yanga, Chen-Yan Zhanga,*, Qian-Jin Wangb, Yun-Zhu Guoa, Chen Donga, Er-Kai Yana, Wen-Jing Liua, Xi-Wang Zhenga, and Da-Chuan Yina,* a


School of Life Sciences, Northwestern Polytechnical University, Xi'an 710072, Shaanxi, PR China b


Email of the corresponding author: Chen-Yan Zhang: [email protected], and Da-Chuan Yin: [email protected].

In biopharmaceutical and food industry applications, the nucleants should preferably be, in addition to capable of enhancing protein crystallization, biocompatible, nontoxic and metabolizable to avoid any unfavorable effects. Cyclodextrins may be a possible choice that can be applied not only in the biopharmaceutical and food industries but also in the structural determination of proteins. In this research, we investigated cyclodextrins, as potential nucleants for protein crystallization. The experimental results confirmed that β-CD and its derivatives showed significantly positive effects. It was found that a larger number of protein molecules attached to the particles usually corresponded to a higher crystallization success rate. More detailed analysis showed that cyclodextrins exhibited the best performance when the protein in solution had the opposite charge than the cyclodextrins. Our results showed that cyclodextrins can be useful as nucleants due to the ease of modifying them to suit the crystallization of different proteins, and they can be explored for use in the mass purification of proteins for the biopharmaceutical industry. Furthermore, the phenomenon discovered in this study pointed toward a way to find new nucleants based on the overall charge of proteins in a solution: the nucleants should preferably be of overall charge of target protein was opposite to the zeta potential of cyclodextrin particle.


2

Page 3 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Utilization of cyclodextrins and its derivative particles as nucleants for protein crystallization Xue-Zhou Yanga, Chen-Yan Zhanga,*, Qian-Jin Wangb, Yun-Zhu Guoa,§, Chen Donga, Er-Kai Yana, Wen-Jing Liua, Xi-Wang Zhenga, Da-Chuan Yina,* a

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

Biotechnology, School of Life Sciences, Northwestern Polytechnical University, Xi’an 710072, Shaanxi, PR China b


KEYWORDS: cyclodextrin, protein crystallization, nucleation, charge

The authors declare no conflict of interest. * To whom correspondence may be addressed. §

Present Address: Chang'an University, Middle-section of Nan'er Huan Road, Xi'an 710064,

Shaanxi, P. R. China Chen-Yan Zhang, Email: [email protected], Tel: +86-29-88460543; Da-Chuan Yin, Email: [email protected], Tel: +86-29-88460254.


3


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 60

ABSTRACT

Finding new nucleants to promote protein crystallization is an important task, especially for purposes other than structural determination. Here, we investigated cyclodextrins and its derivative particles, as potential nucleants for protein crystallization. β-cyclodextrin (β-CD) and its derivatives (including p-toluenesulfonyl-β-cyclodextrin (PTCD), polymer-β-cyclodextrin (PCD),

Mono-(6-(1,6-hexamethylenediamine)-6-deoxy)-β-Cyclodextrin

(MHCD)

and

Mercapto-β-cyclodextrin (MCD)) were used as nucleants. The experimental results confirmed that β-CD and its derivatives showed significantly positive effects, promoting protein crystallization and improving crystal quality. A larger number of protein molecules (including lysozyme, catalase, subtilisin A VIII, concanavalin A VI, α-chymotrypsinogen, proteinase K, cellulase, papain, glucose isomerase, hemoglobin and ribonuclease A XII) attached to the particles usually corresponded to a higher crystallization success rate. More detailed analysis showed that cyclodextrins exhibited the best performance when the overall charge of protein in solution was the opposite to zeta potential of the cyclodextrins particle. Our results showed that cyclodextrins can be useful as nucleants due to the ease of modifying them to suit the crystallization of different proteins, and they can be explored for use in the mass purification of proteins for the biopharmaceutical industry. Furthermore, the phenomenon discovered in this study pointed toward a way to find new nucleants based on the overall charge of proteins in a solution: the nucleants should preferably be of the opposite between overall charge of target protein and zeta potential of cyclodextrin particle.


4

Page 5 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1. INTRODUCTION Protein crystallization is a highly useful process in that it cannot only prepare protein crystals for the structural determination of protein molecules (one of the prevailing methods to obtain high resolution molecular structures of proteins1) but also provide highly purified proteins for various purposes2 including biopharmaceuticals3, the food industry4, chemical agents5, catalysis6, and environmental engineering7. Since the crystallization of a protein is frequently difficult, the development of methods to enhance protein crystallization is always important8-10. To succeed in crystallization, the first step is to achieve nucleation11. Nucleation directly from a solution, i.e., homogeneous nucleation, must overcome a high energy barrier. To reduce this energy barrier, seeding and heterogeneous nucleation are often used to promote protein crystallization12. In the investigation of heterogeneous nucleation for protein crystallization, many efforts have been made to find effective nucleants that can promote the nucleation of protein crystals. Numerous nucleants have been studied, including dried seaweed13, horse hair13, cellulose13, hydroxyapatite13, polystyrene nanospheres14, micromica15, chlorite15, porous silicon16, mesoporous bioactive gel-glass17, porous polystyrene-divinylbenzene microspheres18, silanized mica19, lipid bilayers20,21, polymeric film22, porous glass substrate23, fluorinated layered silicate24, colloidal graphenes25, nanowrinkled substrate26, and so on. McPherson originally reported mineral substrate nucleants27, these mineral substrates were based on the hetero-epitaxial growth that was distinct from above methods, also Sugahara et al. developed microporous zeolite as the hetero-epitaxial nucleants28,29. Most of these nucleants have proven effective, and some have been even commercialized16,30. The past investigation of heterogeneous nucleants has mainly focused on helping to obtain high-quality protein crystals for structural determination. In biopharmaceutical and food industry


5


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 60

applications, the nucleants used during the process of crystallization should preferably be, in addition to capable of enhancing protein crystallization, biocompatible, nontoxic and metabolizable to avoid any unfavorable effects. Though there already have been nucleants that may partially meet the above requirements, more efforts to find suitable nucleants and studies on related mechanisms are still helpful, so that the utilization of nucleants can be more rational and more effective in practical applications. In searching for new nucleants for protein crystallization, we noticed that cyclodextrins may be a possible choice that can be applied not only in the biopharmaceutical and food industries but also in the structural determination of proteins. Cyclodextrins are a family of compounds that are widely used in the pharmaceutical31 and food industries32,33. “Cyclodextrins” is a general term for cyclic oligosaccharides. The structure of cyclodextrins is unique, resembling a thick-walled bucket with external hydrophilic edges and a hydrophobic central cavity34. The central cavity is capable of entrapping various hydrophobic and amphiphilic guest molecules to form noncovalent complex35. Due to its remarkable encapsulation properties, it has potential applications in many fields. In addition to their applications in food and pharmaceutical industries, the cyclodextrins are also utilized in chemistry36, chromatography37, catalysis38, biotechnology39, agriculture40, and environmental engineering41. As one of the most widely used cyclodextrins, β-cyclodextrin (β-CD) consists of seven α-1, 4 linked glucose units. It can incorporate hydrophobic residues or partially unfolded intermediates of protein to enhance protein refolding or stabilization, and protein structure is not affected by β-CD42, so it is widely used in protein sample preparation43-47. Methylated β-cyclodextrin has been also used in extracting detergents from a detergent-membrane protein complex to obtain protein 2D crystal48. It has been proved to improve the protein reconstitution


6

Page 7 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


due to detergent complexation by cyclodextrins49. β-CD can be chemically modified to obtain different derivatives with various functional groups. Since it is widely used, commercial β-CD derivative products are easily available. The properties of β-CD and its derivatives have also been thoroughly studied, making necessary information conveniently available. Based on the above considerations, we selected β-CD and its derivatives particle as possible nucleants for protein crystallization and investigated the effect of these cyclodextrins on protein crystallization. The possible mechanism and potential applicability are also discussed.


7


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 60

2. MATERIALS AND METHODS 2.1. Materials As listed in Table 1, eleven commercial proteins were used in this study. All of the proteins were dissolved in their corresponding buffers (in Table 1) without further purification. Sodium acetate, sodium chloride and HEPES sodium were bought from Beijing Chemical Factory (Beijing, China). The structure of β-CD was schematically shown in Fig. 1 a. Apart from β-CD (obtained from Beijing Solarbio Science & Technology Co., Ltd., Beijing, China), other 6 derivatives (Methylated-β-cyclodextrin (MECD), p-toluenesulfonyl-β-cyclodextrin (PTCD), Mercapto-β-cyclodextrin (MCD), Mono-(6-ethanediamine-6-deoxy)-β-Cyclodextrin (MOCD), Mono-(6-(1,6-hexamethylenediamine)-6-deoxy)-β-Cyclodextrin

(MHCD),

and

Hydroxypropyl-β-cyclodextrin (HPCD)) of β-CD and a mixture, polymer-β-cyclodextrin (PCD), were used in this study. All of these derivatives were purchased from Zhiyuan Biotechnology Co., Ltd. (Shandong, China). The structures of the derivatives are shown in Figs. 1 b1- b6. The crystallization screening kit IndexTM used in this study was obtained from Hampton Research Co. Ltd (Aliso Viejo, USA). The Crystal Clear Sealing Tape was from Hampton Research Co. Ltd, and the Intelli-plate 96-2 was obtained from Art Robbins Instruments Co. Ltd (Sunnyvale, CA).


8

Page 9 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 1. Proteins and their buffers used in this study

100 mM sodium acetate, pH 4.6

Initial concentration (mg/ml) 20a

Seikagaku

Cat.

25 mM HEPES sodium, pH 7.0

10

Sigma-Aldrich

Subtilisin A VIII

Sub.


20

Sigma-Aldrich

Concanavalin A VI

Con.


10

Sigma-Aldrich

α-Chymotrypsinogen II

Chy.


20

Sigma-Aldrich

Proteinase K

Prk.


20

Sigma-Aldrich

Cellulase

Cel.


20

Sigma-Aldrich

Papain

Pap.


14

Sigma-Aldrich

Glucose Isomerase

Glu.


7

Hemoglobin

Hem.


20

Sigma-Aldrich

Ribonuclease A XII

Rib.


20

Sigma-Aldrich

a

Proteins

Abbreviation

Buffers

Lysozyme

Lys.

Catalase

Suppliers

Hampton Research

indicate lysozyme concentration in the crystallization screening study.

Figure 1. The structure of β-CD and their derivatives used in this study. (a1), The chemical structure of β-CD,


9


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 60

(a2), schematic drawing of the cyclodextrin cylinder. (b1) - (b6), the chemical structure of the derivatives PTCD, MHCD, MCD, MECD, MOCD, and HPCD. PCD is polymer of β-CD, the number of monomer is uncertain, so its structure is not shown in Figure 1.

2.2. Crystallization experiments 2.2.1. Crystallization reproducibility study Due to the reproducibility issue50 often encountered in protein crystallization, reproducibility study51, i.e., repetition of the same crystallization experiment to check the crystallization success rate, is employed to examine the effect of a given factor on protein crystallization. Even subtle effects can be found via this method. Lysozyme was selected as a model protein for the reproducibility study. Sitting drop vapor diffusion method was used. To avoid weighing errors during allotment, we first dispensed β-CD and its derivatives in deionized water to obtain dispersing solutions at different concentrations (including 1 mg/ml, 5 mg/ml, 10 mg/ml, 50 mg/ml and 100 mg/ml). Two microliters of the solution was added to each of the 96 wells of the crystallization plate and allowed to dry at 333 K for 3 h, so that different amounts (0.002 mg/well, 0.01 mg/well, 0.02 mg/well, 0.1 mg/well and 0.2 mg/well) of β-CD and its derivatives would remain in the wells. Lysozyme powder was weighed using a high-precision balance (Sartorius BS124S, Gottingen, Germany) and dissolved in a sodium acetate buffer (100 mM, pH 4.6) to obtain lysozyme solutions at initial concentrations of 30 and 40 mg/ml. The lysozyme solutions were then separately mixed with 100 mg/ml NaCl aqueous solution at a volume ratio of 1:1 to obtain crystallization solutions with initial concentrations of 15 mg/ml and 20 mg/ml for lysozyme and 50 mg/ml for NaCl. After mixing, 2 µl of the crystallization solution was dispensed into each of the 96 wells of the crystallization plate using a Crystal Gryphon LCP protein crystallization robot


10

Page 11 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(Art Robbins Instruments, Sunnyvale, CA). The reservoir solution for each well was 80 µl of NaCl solution (100 mg/ml). The crystallization plate was sealed immediately with Crystal Clear Sealing Tape after the solutions were dispensed. The crystallization plates were placed in a home-made temperature controlled chamber51 at 293 K for 48 h. After incubation, the crystallization plates were checked by an automated crystal image reader (XtalFinder XtalQuest Inc., Beijing, China). Due to the resolution limit of the microscope, we defined that a successful crystallization (or a hits) is the one that the crystallization droplet yielded distinguishable crystals under the image reader. The number of droplets that produced lysozyme crystals was obtained, and the crystallization success rate, i.e., the ratio of the number of droplets that produced crystals to the total number of droplets, was then calculated. Based on the reproducibility study, the types of cyclodextrins that showed satisfactory effects on promoting protein crystallization were selected for further study. 2.2.2. Crystallization screening study A crystallization screening study examines crystallization probability by mixing a protein solution with the precipitant solutions from a crystallization screening kit. The number of droplets yielding crystals (hits) shows the crystallization probability for a specific protein. Such a study can compare crystallization screening performance under different conditions. Based on the results of the crystallization reproducibility study, the cyclodextrins including β-CD, MHCD, PCD, PTCD and MCD were selected for the crystallization screening study to examine their ability to promote crystallization in crystallization screening. These cyclodextrins were dispensed into deionized water at the concentrations (5 mg/ml for β-CD, MHCD and PCD, 10 mg/ml for PTCD and MCD) that showed the best performance in the crystallization reproducibility study.


11


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 60

The sitting drop vapor diffusion method was also used in this part. In this experiment, the cyclodextrins were dispensed into the crystallization plates and treated as described in the reproducibility study. Protein solution was prepared at the initial concentrations (as listed in Table 1). The crystallization droplets were set up by mixing 1 µl protein solution with 1 µl precipitant solution from the crystallization screening kit IndexTM, and 80 µl of the precipitant solution was used as the reservoir solution for the corresponding crystallization droplets. The crystallization plates were placed in the temperature controlled chamber at 293 K for 48 h, and then the crystallization droplets were examined as described in the section on the crystallization reproducibility study. To examine the situation in longer time range we used two proteins (lysozyme and glucose isomerase) as the examples for the testing. We checked the hits after adding β-CD, PCD, MHCD, PTCD and MCD for 2 days, 5 days, 10 days and 15 days. 2.3. X-ray diffraction experiment To examine the effect of adding cyclodextrins on the quality of the crystals, X-ray diffraction experiments were carried out on crystals of two arbitrarily selected proteins (lysozyme and proteinase K) grown with and without adding cyclodextrins. Lysozyme was dissolved in 100 mM sodium acetate pH 4.6, the initial lysozyme concentration was 40 mg/ml, the crystallization agent was 80 mg/ml NaCl. The crystals were grown in the presence of three types of cyclodextrins (β-CD, PTCD and MHCD) at 293 K for 48 h. In the case of proteinase K, the initial concentration of proteinase K was 20 mg/ml in 100 mM Tris-HCl at pH 8.5, the crystallization agent was 158.57 mg/ml (NH4)2SO4. The cyclodextrin used was MHCD. Crystals with similar size (200-400 µm) in cyclodextrin group and control were harvested by using nylon CryoLoop (Hampton Research Co. Ltd, USA) for X-ray diffraction testing. All data was collected at 100 K in nitrogen stream by using X-ray single-crystal diffraction system


12

Page 13 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(MarResearch GmbH Norderstedt, Germany). HKL200052 and CCP453 were used to process and scale the diffraction data. The method for calculating of B factor value was Wilson plot. 2.4. Particle size and zeta potential measurements Monitoring the particle size and the zeta potential can give information on the interaction between the protein molecules and the cyclodextrin particles in solution, which may affect the protein crystallization process. Therefore, we studied the particle size and zeta potential of the solution under solution conditions identical to the ones used in the crystallization experiment. To test the difference in the particle size and zeta potential with and without the addition of cyclodextrins, three sets of solutions were prepared: (1) lysozyme (15 mg/ml); (2) β-CD and its derivatives including β-CD (5 mg/ml), PTCD (10 mg/ml), PCD (5 mg/ml), MHCD (5 mg/ml) and MCD (10 mg/ml); and (3) a mixture of lysozyme and cyclodextrin at the same final concentrations. According to the instruction of the products, the solubility of β-CD, CD, MHCD, PTCD and MCD in H2O at 293 K is 14.3 mg/ml, 100 mg/ml, 150 mg/ml, 0.4 mg/ml and 10 mg/ml, respectively. Therefore β-CD, PCD, MHCD and MCD can be dissolved completely at the concentrations used in the study (5 mg/ml for β-CD, PCD and MHCD, 10 mg/ml for PTCD and MCD), except for PTCD. All of the above solutions were prepared using a 100 mM sodium acetate buffer (pH 4.6) containing 50 mg/ml NaCl. A dynamic light scattering (DLS) instrument (Zetasizer Nano ZS (Malvern Instruments Ltd., Malvern, Worcestershire, UK)) was used for the measurement. The solutions were measured immediately after preparation, and 200 µl solution was used for this measurement. The same solutions but without NaCl were prepared to measure the zeta potential to account for its effect on electrophoresis. The same instrument (Zetasizer Nano ZS) was used for this measurement. The volume of solution used was 1 ml.


13


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 60

3. RESULTS 3.1. Crystallization reproducibility results Before the crystallization reproducibility experiments, we carried out an initial check of the effect of adding cyclodextrins on lysozyme crystallization, and it was found that some types of cyclodextrins (MECD, HPCD, and MOCD) were not satisfactory in promoting the crystallization, while others (β-CD, PTCD, PCD, MHCD, and MCD) showed a positive effect, increasing the chance to obtain protein crystals. Hence, in both the crystallization reproducibility and screening studies, we chose β-CD, PTCD, PCD, MHCD, and MCD as the nucleants. In the crystallization reproducibility study, we used different amounts of β-CD and its derivatives in each crystallization droplet. Five different amounts of cyclodextrins, i.e., 0.002 mg/well, 0.01 mg/well, 0.02 mg/well, 0.1 mg/well and 0.2 mg/well, and two lysozyme concentrations (15 and 20 mg/ml after mixing) were used in the study. The results are shown in Figure 2. The crystallization success rate was clearly dependent on the amount of cyclodextrins (Figs. 2a and 2b). It should be pointed out that, though the solution of cyclodextrins may appear clear and transparent, but to some extent the cyclodextrin particles in the solution can be still considered as a kind of solid state material that functions normally as heterogeneous nucleates, because the size is usually more than several hundreds of nanometers, much larger than that of the protein molecules. In all cases, there seemed to be an optimal amount of cyclodextrins at which the crystallization success rate reached a maximum. The best amounts of cyclodextrins were 0.01 mg/well (i.e., 5 mg/ml in the droplets) for β-CD, PCD, and MHCD and 0.02 mg/well (i.e., 10 mg/ml in the droplets) for PTCD and MCD. At higher or lower amounts of cyclodextrins, the crystallization success rate would be lower than the optimal one. One more phenomenon that must be addressed is that upon adding more cyclodextrins, the


14

Page 15 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


crystallization success rate would ultimately be decreased to a level even lower than the control. The initial concentration of lysozyme also showed some effect on the crystallization success rate. At different initial lysozyme concentrations, the increase in the crystallization success rate at the same amount of cyclodextrins was different. To see the overall effect of adding cyclodextrins, we averaged the normalized crystallization success rate at two initial lysozyme concentrations (15 and 20 mg/ml) and have summarized the results in Fig. 2c for different cyclodextrins. A similar trend to the one observed in Figs. 2a and 2b can also be found in Fig. 2c. At the same time, we noticed that different cyclodextrins showed different performance in enhancing crystallization. To check the optimal performance of each cyclodextrin, we averaged the best normalized crystallization success rate of each cyclodextrin, and these values are displayed in Fig. 2d. It can be seen that the best performance varied with the cyclodextrin type, and the effect of PTCD showed the best performance (up to approximately 2 times the control) in enhancing lysozyme crystallization. The crystallization success rate for different cyclodextrins can also be different. For example, the best crystallization success rate using PTCD was 1.55 times higher than the best rate using MHCD.


15


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 60

Figure 2. Normalized crystallization success rate of lysozyme using different amounts of β-CD and its derivatives in the reproducibility study. Two initial concentrations (after mixing) of lysozyme were used: (a) 15 and (b) 20 mg/ml, and the concentration of NaCl was 50 mg/ml. (a) 15 and (b) 20 mg/ml, and the concentration of NaCl was 50 mg/ml. (c) the average normalized crystallization success rate, (d) best normalized crystallization success rate. Error bar was the standard deviation value. Welch’s t-test was applied for the comparison. Two-tailed test was used. The data fit to Gaussian distribution which was tested by K-S test, the variation of two groups was unequal which was tested by F-test, so we used Welch’s t-test for the statistical analysis.

To further verify the effect of these cyclodextrins on protein crystallization, we used an extremely low initial concentration of lysozyme (2.5 mg/ml after mixing) for testing. The NaCl concentration was 50 mg/ml (after mixing). No crystals could be obtained at such a low lysozyme concentration in the control, while crystals could be found in droplets containing β-CD, PTCD, and MCD. This test further confirmed that the presence of β-CD and its derivatives was


16

Page 17 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


beneficial for protein crystallization. We also compared the crystal morphology of lysozyme with and without adding cyclodextrins. Figure 3 showed typical images of the crystal morphology with different amounts of different cyclodextrins. A trend in crystal number can be observed that as the amount of cyclodextrins increased, the number of crystals increased, but simultaneously, the crystal size decreased. Observing the crystal size and crystal number further confirmed that the addition of β-CD and its derivatives facilitated the crystallization of lysozyme.

Figure 3. Example images of crystallization droplets with different amounts of β-CD and its derivatives. The amount of β-CD, PTCD, PCD, MHCD or MCD in each crystallization droplet was 0 mg/well, 0.002 mg/well, 0.01 mg/well, 0.02 mg/well, 0.1 mg/well and 0.2 mg/well, respectively. Lysozyme and NaCl concentrations were 20 and 50 mg/ml after mixing. It should be noted that the cyclodextrin was easy to be aggregated, even when the concentration of β-CD was 1 mg/ml (0.002 mg/well) (Fig. S1), and these aggregates were difficult to


17


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 60

reduce or remove54.

3.2. Crystallization screening results Eleven commercial proteins (as listed in Table 1) were used in the crystallization screening study. Figure 4 compares the crystallization hits with and without adding the cyclodextrins. All of the results (Figs. 4 a1 - e1, and Figs. 4 a2 - e2) showed that the addition of the tested cyclodextrins exhibited statistically positive effects on promoting crystallization. Among the cyclodextrins tested, β-CD showed the greatest effect, and MHCD and PTCD took the second place. Some crystals could only be obtained when using β-CD and its derivatives, for example, ribonuclease A XII and subtilisin A VIII crystal can only be obtained using β-CD, PTCD, PCD, and MCD, and papain crystal can only be obtained using β-CD, PCD, MHCD and MCD. In the examination of crystallization with cyclodextrins in longer time ranges, we have noticed that the crystallization hits increased upon incubating for longer time in both groups with and without cyclodextrins. In the figure of the normalized hits (Fig. S2) using the hits in the control group as the basis (i.e., the normalized hits in the control group is 1), the normalized hits in the cyclodextrins group decreased with the time, showing that the crystallization (nucleation) occurred later or slower in the control group than in the cyclodextrins group. Noticeably the crystallization hits reached similar level in both groups in the cases with PCD, MHCD and MCD. However, the crystallization hits in the cyclodextrins groups were still higher or equivalent to those of the control groups. And in the cases with β-CD and PTCD, the crystallization hits in the cyclodextrin groups were still significantly higher than in the control groups in a prolonged time period. These results confirmed that cyclodextrins can facilitate crystallization of the tested proteins. The improvement in crystallization screening hits depended not only on the types of


18

Page 19 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


proteins but also on the types of cyclodextrins. Adding the same cyclodextrins to different protein crystallization droplets would result in different improvement effects. For example, adding PTCD to the crystallization solutions of different proteins can result in hits improvement 177% (Fig. 4 b1). Adding different cyclodextrins to the crystallization solution of the same protein would also result in different improvement effects. For example, for proteinase K, the hit improvement was 200% using MHCD (Fig. 4 d1), 193% using PCD (Fig. 4 c1), and only 96% using MCD (Fig. 4 e1). We also compared the unique crystallization hits between cases with added cyclodextrins and the control, summarized from all five sets of experiments using different cyclodextrins. The results showed that the total of unique crystallization hits using cyclodextrins was 197%, p = 0.00003 < 0.001, (see Fig. 4 f1 - f2). The most prominent example was hemoglobin: the number of unique crystallization hits was 1 in the control, while in the case with added cyclodextrins, the number of unique crystallization hits was 12.


19


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 60

Figure 4. Comparison of crystallization screening hits with and without adding cyclodextrins. β-CD and its derivatives were used: (a1) and (a2) are β-CD (p = 0.00003 < 0.001); (b1) and (b2) are PTCD (p = 0.0006 < 0.001); (c1) and (c2) are PCD (p = 0.0002 < 0.001); (d1) and (d2) are MHCD (p = 0.00009 < 0.001); (e1) and (e2) are MCD (p = 0.0013 < 0.001); (f1) comparison of the number of unique hits with and without adding cyclodextrins; (f2) comparison of the average normalized unique hits with and without adding cyclodextrins (p = 0.00003 < 0.001). All of the results showed a positive effect, promoting protein crystallization, of all five types of cyclodextrins. Error bar was the standard deviation value. Non-parametric method Kolmogorov-Smirnov test was applied for the statistical analysis, two-tailed test was chosen.


20

Page 21 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


We also summarized the unique crystallization hits found only in the control or in the group with cyclodextrins. Table 2 shows the results. We can see that there were always some unique crystallization conditions that appeared only in the control or in the group with a certain type of cyclodextrins. Apparently, the combination of these unique crystallization conditions can further increase the chance to obtain crystals.

Table 2. Unique crystallization conditions found only in the control or in the experimental group with the addition of a certain type of cyclodextrins control

β-CD

PTCD

PCD

MHCD

MCD

Con.

NULL (21)a

NULL (21)

34, 36, 50 (24)

NULL (21)

40 (22)

93 (22)

Glu.

32 (2)

61 (2)

15, 17, 63 (4)

11, 12, 36 (4)

NULL (1)

NULL (1)

Prk.

62, 84, 93 (5)

39, 46, 47, 51, 53, 55, 92 (9)

20, 56, 57, 58 (6)

NULL (2)

NULL (2)

NULL (2)

Chy.

1, 3, 23, 29 (5)

40, 42, 43, 79 (5)

9, 34, 80 (4)

14, 47, 54, 59 (5)

44 (2)

NULL (1)

Cat.

56, 95 (3)

5 (2)

3, 9, 35 (4)

16 (2)

6, 31, 66 (4)

24, 26, 27, 29, 32, 33, 38, 58 (9)

Lys.

94 (1)

NULL (0)

85, 86, 91 (3)

NULL (0)

67, 69 (2)

NULL (0)

Hem.

86 (1)

21, 41, 43 (3)

33, 54, 94, 95 (4)

63 (1)

11, 67 (2)

55 (1)

Rib.

NULL (0)

6, 10, 58, 61 (4)

91 (1)

37 (1)

NULL (0)

62, 87 (2)

Cel.

68, 84, 94 (3)

39 (1)

NULL (0)

NULL (0)

17, 86 (2)

NULL (0)

Pap.

NULL (0)

5, 6 (2)

NULL (0)

66 (1)

11, 90 (2)

54 (1)

Sub.

NULL (0)

43 (1)

18 (1)

36, 48 (2)

31 (1)

38 (1)

Note: the number in the table was the serial number of the precipitant solution from the crystallization screening kit IndexTM. a

indicate total number of conditions providing crystals including common conditions in a parenthesis.

The crystal morphology showed that the addition of cyclodextrins can indeed affect protein


21


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 60

crystallization. Figure 5 shows some examples of crystal images obtained under the same crystallization condition (i.e., the same serial number of the precipitant from the crystallization screening kit) using different types of cyclodextrins. From the figure, we can see not only that the addition of cyclodextrins can affect the probability of obtaining crystals but also find that the crystal morphology could be affected. For example, concanavalin A crystals showed needle-like morphology when grown without adding MHCD, while the crystals became isometric with well-defined facets when MCD was added.

Figure 5. Examples of crystallization droplets images with and without the addition of cyclodextrins. The images show droplets of concanavalin in H7 (No. 91 from IndexTM: 0.15 M DL-Malic acid pH 7.0, 20% w/v Polyethylene glycol 3350) of the screening kit IndexTM, glucose isomerase in E6 (No. 54 from IndexTM: 0.05 M Calcium chloride dehydrate, 0.1 M Bis-Tris pH 6.5, 30% v/v Polyethylene glycol monomethyl ether 550), proteinase K in C9 (No. 33 from IndexTM: 1.1 M Sodium malonate pH 7.0, 0.1 M HEPES pH 7.0, 0.5% v/v Jeffamine ® ED-2001 pH 7.0), chymotrypsinogen in G3 (No. 75 from IndexTM: 0.2 M Lithium sulfate monohydrate, 0.1 M Bis-Tris pH 6.5, 25% w/v Polyethylene glycol 3350) and lysozyme in B3 (No. 15 from IndexTM: 0.1 M HEPES pH 7.5, 0.5 M Magnesium formate dihydrate).


22

Page 23 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


3.3. Comparison of crystal quality with and without cyclodextrin It has been shown that the success rate of protein crystallization can be increased by adding the cyclodextrins. To test whether the crystal quality can be also improved with addition of cyclodexin, lysozyme and proteinase K were used for the X-ray diffraction testing. Since β-CD, PTCD and MHCD showed the best performance in increasing the success rate of crystallization, we used them in the crystal quality comparison. The X-ray diffraction results are shown in Figure 6 and Figure 7, the detail X-ray diffraction data are shown in Table S1 and Table S2. It can be seen that the resolution limit of lysozyme crystals was improved by using β-CD, PTCD and MHCD. While the improvement in the resolution limit was different by using different cyclodexins. For PTCD, the resolution limit was improved from 2.23 Å in the control to 2.06 Å in the PTCD group; For MHCD, the resolution limit was improved from 2.23 Å in the control to 1.88 Å in the MHCD group (p = 0.045 < 0.05). In Figure 6 (a2), the average resolution limit was improved from 2.23 Å in the control to 1.94 Å in the cyclodexin group (p = 0.032 < 0.05). In Figure 6 (b) and (c), the B factor and the mosaicity were also improved in the cyclodexin group compared with that in the control group, however there were no statistical significance. According to the above result, the quality of lysozyme crystal showed the most significant improvement by using MHCD. We further tested the effect of adding MHCD on the quality of proteinase K crystal. The resolution limit was improved from 2.29 Å in the control to 1.81 Å in the MHCD group (p = 0.015 < 0.05). According to the tested results, we can say that the quality of crystal can be improved with addition of cyclodexins.


23


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 60

Figure 6. Comparison of the quality of lysozyme crystals grown with and without adding cyclodextrins. (a1) (a2) are the resolution limit of lysozyme crystals grown with and without cyclodextrins (including β-CD, PTCD and MHCD). In (a1), p = 0.045 < 0.05 for MHCD, in (a2), p = 0.032 < 0.05; (b1) - (b2) and (c1) - (c2) are the B factor and the mosaicity of lysozyme crystals with and without adding cyclodextrins, respectively. Error bar was the standard deviation value. Two-tailed test was used. The data fit to Gaussian distribution which was tested by K-S test, the variation of two groups was unequal which was tested by F-test, so we used Welch’s t-test for the statistical analysis.


24

Page 25 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 7. Comparison of the quality of proteinase K crystals grown with and without adding cyclodextrins (MHCD). (a) the resolution limit of proteinase K crystals with and without adding MHCD (p = 0.015 < 0.05); (b) and (c), the B factor and the mosaicity of proteinase K crystals with and without adding MHCD, respectively. Error bar was the standard deviation value. Two-tailed test was used. The data fit to Gaussian distribution which was tested by K-S test, the variation of two groups was equal which was tested by F-test, so we used Student’s t-test for the statistical analysis.

3.4. Particle size and zeta potential measurements DLS and zeta potential measurements are useful tools for studying the interactions among the particles in the solution and to predict the initial nucleation process during protein crystallization. In this research, we measured the particle size in solutions of proteins, solutions of cyclodextrins, and mixed solutions of protein and cyclodextrins. The solution conditions were identical to the ones used in the crystallization reproducibility experiments. The protein used was lysozyme, and all five types of cyclodextrins were tested in these measurements. Figure 8 shows the results of particle size measurements using DLS. In the protein crystallization solution of lysozyme without cyclodextrins, the particle size was mostly at approximately 2.2 nm - 2.5 nm, which was apparently the size of lysozyme monomer. There was another peak at approximately 590 nm, indicating that there were some clusters in the solution. In a solution of cyclodextrins, there was usually a peak ranging from 377 nm (β-CD) to 1304 nm


25


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 60

(MCD). The size was apparently not the same as for cyclodextrin monomers, but that of aggregated clusters of the cyclodextrins. It has been reported that cyclodextrin can form aggregates in water55. By using Cryo-TEM and DLS experiment, Bonini et al.56 confirmed that β-CD aggregate formed in the water at room temperature, which was dependent on the concentration of β-CD, the diameter of β-CD aggregate particle was about 100 nm at lower concentration, while this diameter would increase significantly at higher concentration. Further, we used DLS to compare the average particle size with and without adding NaCl into cyclodextrin solution, it showed that the average particle size slightly increased (Fig. S3). However, the mechanism remains unclear. After adding the cyclodextrins to the protein crystallization solution, we can find that the particle size in the solution becomes larger than for either the cyclodextrins or the proteins (the size increased to the range between 652.8 nm - 2153 nm), indicating that the cyclodextrins and protein molecules interacted to form new and larger clusters. Due to the DLS results in Figure 8 were shown only in intensity, which may emphasize the peaks in larger particles, so we further added the percentage of number of particle size in supplementary part (Fig. S4). The result in Figure S4 was similar with that of Figure 8. Notably, it can be seen that, among the cyclodextrins, PTCD and MCD showed different features from the others in the DLS size measurement. The size of the particles in their own solutions was larger than other cyclodextrins, and the particle size in the solutions with protein was also larger than for other cyclodextrins. At the same time, the lysozyme monomer peak disappeared in the solutions with PTCD and MCD. These results indicated that more lysozyme molecules were adsorbed onto PTCD or MCD particles in the solution. PTCD and MCD also showed the most prominent effect in increasing the crystallization success rate of lysozyme, as shown in Fig. 2.


26

Page 27 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 8. Particle size measurement results in the solutions of protein, cyclodextrins, and protein with added cyclodextrins. The particle size distributions in the solutions upon adding β-CD, PTCD, PCD, MHCD and MCD are shown in (a), (b), …, (e), respectively. Solution 1 (the red dotted curves): crystallization solution of lysozyme. Concentration of lysozyme was 15 mg/ml, concentration of NaCl was 50 mg/ml. Solution 2 (the green dashed curves): cyclodextrin solution (concentration of β-CD, PCD, and MHCD was 5 mg/ml; for PTCD and MCD: 10 mg/ml; for NaCl: 50 mg/ml). Solution 3 (the purple solid curves): solution with lysozyme and cyclodextrins mixed.

Since the adsorption of lysozyme molecules to the cyclodextrins should be correlated with the surface potential of the particles, we measured the zeta potential of the particles in the above solutions. Figure 9 shows the measurement results. All measurement results showed that the zeta


27


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 60

potential of the solution with mixed lysozyme and cyclodextrins was in between the values for the solutions with lysozyme and cyclodextrins alone. The zeta potential of lysozyme in the solution was mostly in the range of 6.3 mV - 6.9 mV, and the zeta potential of the cyclodextrins varied in a much larger range (from -2.1 to 7.7 mV). Two cyclodextrins in the solution were negatively charged (β-CD and MCD), and one (MHCD) was positively charged. The rest (PTCD and PCD) were neutral. All of the cyclodextrins were dissolved in 100 mM sodium acetate buffer pH 4.6. For the β-CD, the CH3COO- combine with -OH of β-CD, so β-CD carried weak negative charge. For MCD, the sulfydryl of the MCD could dissociate to -S- and H+, thus MCD residue carried negative charge.


28

Page 29 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 9. Zeta potential measurement results in solutions of protein, of cyclodextrins, and of protein with the addition of cyclodextrins. The concentration of lysozyme was 15 mg/ml; the concentration for β-CD, PCD, and MHCD was 5 mg/ml; the concentration for PTCD and MCD was 10 mg/ml. Error bar was the standard deviation value.


29


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 60

4. DISCUSSIONS 4.1. General process of crystallization when cyclodextrins were added to the crystallization solution Nucleation is the first step of crystallization. Crystallization without any solid-liquid interface begins nucleation from the solution, which is known as homogeneous nucleation. In practical protein crystallization, crystallization in most cases tends to nucleate at a solid-liquid interface (known as heterogeneous nucleation), such as the wall or bottom of the crystallization wells. When there are crystal seeds or heterogeneous particles in the solution, epitaxial growth or heterogeneous nucleation of the protein crystal may occur at the surface of the seeds or the particles. In this study, we used cyclodextrins in the solution. According to the DLS measurement, there were cyclodextrin particles suspended in the solution. The size of the cyclodextrin particles ranged between 377.7 nm to 1304 nm, much larger than the size of the lysozyme molecules in the solution (approximately 2.2 - 2.5 nm). After the addition of the cyclodextrins to the crystallization solution, we observed a clear increase in particle size in the solution, indicating that the lysozyme molecules were adsorbed onto the cyclodextrin particles. Based on this observation, we suggested that adsorption of the protein molecules would result in an increase of concentration at the surface of the cyclodextrin particles, where nucleation will be easier to promote crystallization of the protein. Figure 10 shows schematically the processes after the addition of cyclodextrins into the crystallization solution. Before cyclodextrin addition, lysozyme molecules were distributed homogeneously in the solution. After the addition of the cyclodextrins, the adsorption of protein molecules began. At the surface of the cyclodextrin particles, the trapped protein molecules formed a relatively higher local concentration than in the bulk solution. Hence, nucleation at the surface will become easier, and crystallization will be


30

Page 31 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


thus promoted. The above mechanism is applicable when the concentration of protein molecules is suitable. At higher protein concentrations, crystallization may occur at various sites including the surface of the suspending particles, the surface of the well, or directly from solution (homogeneous nucleation). At protein concentrations too low to succeed in crystallization, crystallization may not occur. The above mechanism is also applicable when the amount of cyclodextrins in the solution is suitable. When the amount of cyclodextrins in the solution is too little, the cyclodextrins are completely dissolved in the solution, and the promoting effect may not be obvious. When the amount of cyclodextrins in the solution is too great, most of the protein molecules will be captured by cyclodextrin particles. In this case, there will not be enough protein in the bulk solution to support crystal growth, and therefore a decrease in the crystallization success rate may be observed. This speculation is in good agreement with our experimental observations, as illustrated in Fig. 2.

Figure 10. Model of the protein molecules adsorbed onto cyclodextrin molecules during crystallization.

4.2. Correlation of adsorption of lysozyme with crystallization success rate Upon further examining our experimental data, we found more detailed information on the relationship between the size change of the particles in the solution and the crystallization success rate. As mentioned in the crystallization reproducibility experimental results section, the effect of adding PTCD and MCD to the solution showed a prominent increase in crystallization


31


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 32 of 60

success rate. It happened that, in the DLS measurement, these two cyclodextrins showed the greatest increase in the particle size after addition into the protein crystallization solution. To further explore whether there existed any relationship between the size increase and crystallization success rate, we summarized the size increase after cyclodextrin addition and the crystallization success rate after adding the corresponding cyclodextrins. Figure 11 shows the results. We can tell that the trend of the crystallization success rate against cyclodextrin type mostly followed the size increase trend against the corresponding cyclodextrins. In other words, we observed that, when the size increase was larger, the crystallization success rate was higher. The size increase of the particles indicated the number of protein molecules attached to the particle. Based on the particle size determined by the DLS, the particle volume can be calculated from its radius, so the volume of lysozyme, cyclodextrin, lysozyme and cyclodextrin mixed particles can be calculated, and then the number of lysozyme molecules attached to the cyclodextrin particles can be calculated by the following formula. It should be noted that this is only an assumption for convenient calculation because in the actual case the size of the cyclodextrin particles may be varied by attachment of protein molecules, or by rearrangement of the aggregation of the cyclodextrin molecules. 4π 4π 4π 3 3 3 3 RCD+Lys. = 3 RCD + n × 3 RLys. ,

(1)

where RCD+Lys. is the radius of the particles in the solution with mixed cyclodextrin and lysozyme; RCD is the radius of the particles in the solution of cyclodextrin; and RLys. is the radius of the lysozyme molecule. Based on the DLS measurement data, we obtained the following numbers of lysozymes attached to different cyclodextrin particles: 5.7 × 108, 3.9 × 109, 3.4 × 108, 1.2 × 108, and 6.7 × 109 corresponding to β-CD, PTCD, PCD, MHCD, and MCD, respectively. We can see that the


32

Page 33 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


results of this calculation matched well with the size increase data, and hence the number of lysozyme molecules attached to the cyclodextrin particles indicated the ability of the cyclodextrins to promote lysozyme crystallization. From these results, we may conclude that the ability to capture lysozyme molecules may indicate the power to promote crystallization.

Figure 11. The correlation between size increase of particle (black dot) and normalized crystallization success rate (red square) upon adding cyclodextrins.

4.3. Correlation of the charges carried by cyclodextrins and proteins with the crystallization screening results As discussed above, lysozyme molecules were adsorbed onto cyclodextrins, and the number of lysozyme molecules adsorbed was correlated with the crystallization success rate. The reason that cyclodextrins can capture protein molecules may originate from the interactions between the protein molecules and the cyclodextrin particles. Based on the measured zeta potential of the particles in the solution, we can see that the particles in the solution were either charged or neutral. The lysozyme in the crystallization solution, however, was positively charged. The charges carried by the particles in the solution maybe affect their interaction. Negatively charged particles can always attract positively charged ones, while particles with the same charged always repel each other. Hence, the difference in the charges carried by the particles and the


33


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 34 of 60

protein molecules may crucially affect the crystallization results. The charge of proteins here was shown in Table S3. The ratio of hits between cyclodextrins and control group was shown in Table S4. For con. (negatively charged), hits ratio was minimum with additional of MCD compared with other cyclodextrins (negatively charged). For glu. (negatively charged), hits ratio was maximal by using MHCD (positively charged), and this value went to minimum by using MCD (negatively charged) compared with other cyclodextrins. For chy. (positively charged), hits ratio was maximum with additional of MCD (negative charged) compared with other cyclodextrins. For cat. (negatively charged), hits ratio went to maximum by using of MHCD (positively charged) compared with other cyclodextrins. While hits was affected by many factors, charge of protein was only one of them, so hits maybe not only related to the charge, but also other factors. To find out if the zeta potential of the cyclodextrin particles affected protein crystallization, we analyzed the charge of the protein molecules and the cyclodextrins from the crystallization screening experiments. To find out if the zeta potential of the cyclodextrin particles affected protein crystallization, we analyzed the charge of the protein molecules and the cyclodextrins from the crystallization screening experiments. Figure 12 shows the results. In Fig. 12a, two groups of comparisons are made. The normalized crystallization hits of negatively and positively charged proteins with the addition of negatively charged cyclodextrins are denoted N-N and N-P, respectively, while the normalized crystallization hits of negatively and positively charged proteins with the addition of positively charged cyclodextrins (it needed to indicate that the charges are not covalently attached to the molecule) are denoted P-N and P-P, respectively. From the comparison, we find that when the charges of the cyclodextrins and the protein molecules were opposite, the best crystallization screening hits were obtained (N-P and P-N showed the highest crystallization hits). Accordingly, we performed


34

Page 35 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


statistical analysis of the comparisons between the crystallization screening hits obtained when the charges were the same (same charge, P-P and N-N together) and when the charges were opposite (opposite charge, N-P and P-N together). The results (Fig. 12b) confirmed that higher crystallization screening hits were obtained when the charges of the cyclodextrins and proteins were opposite than when the polarities were the same, and the difference was significant. From the statistical analysis, we also noticed that the crystallization hits were higher with the addition of cyclodextrins even if the charges of the cyclodextrins and the proteins were the same. We consider this phenomenon reasonable because the capture of protein molecules can still occur even if the zeta potential of the cyclodextrin particles is the same as the overall charge of proteins. The overall charge only showed a net charge on the particles. It is probable that there is a distribution of different charges around the surface of the particles. The Zeta potential shows the potential difference between the bulk solution and the stationary fluid layer of the particles in the solution, but it cannot tell the exact distribution of the charges on the surface of the particles. As an example, PTCD showed a neutral Zeta potential in the solution. However, PTCD has the negatively charged sulfonyl group which is located at the surface of the molecules. Hence, this molecule can show attraction to positively charged proteins. The crystallization success rate of lysozyme with the addition of PTCD showed a clear increase compared with the control (Fig. 2), and the size increase of PTCD when it was added to lysozyme solution was high. Both facts confirmed that the detailed distribution of charges carried by the particles can also affect the crystallization of proteins.


35


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 36 of 60

Figure 12. Correlation between particle charge and crystallization screening hits. (a) The crystallization screening hits in different groups with cyclodextrins and proteins having the same or opposite charge. N-N: negatively charged cyclodextrin with negatively charged proteins; N-P: negatively charged cyclodextrin with positively charged proteins; P-P: positively charged cyclodextrin with positively charged proteins; P-N: positively charged cyclodextrin with negatively charged proteins. Comparison between control and N-P: p = 0.028 < 0.05. (b) Summarized comparison of crystallization screening hits between cases with cyclodextrins and proteins having the same or opposite charge. Comparison between control and same charge: p = 0.009 < 0.01. Comparison between control and opposite charge: p = 0.0046 < 0.01. Error bar is the standard deviation value of all data in the group. Two-tailed test was used. The data fit to Gaussian distribution which was tested by K-S test, the variation of two groups was unequal which was tested by F-test, so we used Welch’s t-test for the statistical analysis.

4.4. Perspectives on using cyclodextrins or similar materials in protein crystallization In the current research, we have shown that cyclodextrins, a class of versatile materials that can be modified by different functional groups, can be applied as variable nucleants for protein crystallization. Due to their biologically friendly nature, cyclodextrins are not only applicable to promoting protein crystallization for the purpose of structural determination of proteins, which is still essential for the study of protein structure and function, but also applicable to protein purification via crystallization. The proteins obtained via crystallization are highly bioactive and of high purity, which will be very helpful for applications in biopharmaceuticals. In the


36

Page 37 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


pharmaceutical industry, obtaining insulin57 is a good example of purification via protein crystallization. The phenomenon found in this study indicated that, to promote protein crystallization, protein molecules should be adsorbed onto the nucleants in the solution. Cyclodextrins have different distributions of charges on the particle surface depending on the characteristics of the functional groups. The charge distribution can affect their behavior in adsorbing protein molecules. Preferably, a cyclodextrin with a positively charged surface should be chosen as the nucleant when a protein shows a negative charge in solution, whereas to aid the crystallization of a positively charged protein, a cyclodextrin with a negatively charged surface should be selected. From the results of the current study, we can also suggest a possible method to explore new target-oriented nucleants. Depending on the electrostatic characteristics of proteins in the solution, we can modify the nucleants (such as cyclodextrins, or other nucleants) to attract the protein molecules, to better promote crystallization. It may also be useful to develop materials that can simultaneously carry negative and positive charges in distinct areas on the surface, which may be more useful in crystallization screening. In the crystallization of a protein with known crystallization conditions (for example, in the mass production of highly purified protein via crystallization), nucleants with specific electrostatic features and other characteristics (like biologically friendly properties) may be more favorable.


37


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 38 of 60

5. CONCLUSIONS In this research, we systematically investigated the possibility of using β-CD and its derivatives (including PTCD, PCD, MHCD and MCD) as nucleants for protein crystallization. The following conclusions were drawn: 1) Both the success rate of crystallization and the quality of the crystals can be promoted by the addition of β-CD and its derivatives; 2) When the zeta potential of cyclodextrin particle and charge of protein molecule was opposite, the crystallization success rate showed the greatest increase.


38

Page 39 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


ACKNOWLEDGEMENT This work was supported by National Natural Science Foundation of China (Grant No. 11202167, U1632126), The Natural Science Foundation of Shannxi Province, China (Grant No. 2016JM3012), FRF (the Fundamental Research Foundation, 3102016ZY039) of NPU in China, China Postdoctoral Science Foundation (Grant No. 2013T60890), The Seed Foundation of Innovation and Creation for Graduate Students in Northwestern Polytechnical University (Grant No. Z2017238), Undergraduate Training Programs for Innovation and Entrepreneurship in China (Grant No. 201710699332), The Special Fund for Basic Scientific Research of Central Colleges, Chang'an University (Grant No. 310831161004). The authors in NPU would also like to thank NPU Graduate Student Innovation Center for the help in providing part of the experimental conditions.


39


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 40 of 60

AUTHOR INFORMATION Corresponding Author Chen-Yan Zhang: Phone: +86-29-88460543. Fax: +81-29-88460543. E-mail: [email protected] 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. C.Y.Z. and D.C.Y. designed research; X.Z.Y., Q.J.W., Y.Z.G., C.D., E.K.Y., W.J.L. and X.W.Z. performed research; X.Z.Y., C.Y.Z., Q.J.W. and D.C.Y. analyzed data; and X.Z.Y., C.Y.Z., and D.C.Y. wrote the paper. Funding Sources This work was supported by National Natural Science Foundation of China (Grant No. 11202167, U1632126) The Natural Science Foundation of Shannxi Province, China (Grant No. 2016JM3012) FRF (the Fundamental Research Foundation, 3102016ZY039) of NPU in China China Postdoctoral Science Foundation (Grant No. 2013T60890) The Seed Foundation of Innovation and Creation for Graduate Students in Northwestern Polytechnical University (Grant No. Z2017238) Undergraduate Training Programs for Innovation and Entrepreneurship in China (Grant No. 201710699332) The Special Fund for Basic Scientific Research of Central Colleges, Chang'an University (Grant No. 310831161004).


40

Page 41 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Notes The authors declare no completing financial interest


41


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 42 of 60

SUPPORTING INFORMATION DESCRIPTION Summarization of the diffraction data of lysozyme crystals with and without cyclodextrin. Summarization of the diffraction data of proteinase K crystals with and without MHCD. The charge of six selected proteins. The ratio of hits between cyclodextrins and control group. Particle size measurement results in the solutions of 1 mg/ml β-CD (red dot line) and 5 mg/ml β-CD (black full line). Normalized number of hits by additional of cyclodextrins for 2 days, 5 days, 10 days and 15 days. Size comparison of cyclodextrin particles in the solution with and without adding NaCl. Number of particle size measurement results in the solutions of protein, cyclodextrins, and protein with additional of cyclodextrins. This material is available free of charge via the Internet at http://pubs.acs.org.


42

Page 43 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


REFERENCES (1) Pareja-Rivera, C.; Cuéllar-Cruz, M.; Esturau-Escofet, N.; Demitri, N.; Polentarutti, M.; Stojanoff, V.; Moreno, A. Cryst. Growth Des. 2017, 17, 135-145 (2) Vekilov, P. G. Prog. Cryst. Growth Charact. Mater. 2016, 62, 136-154. (3) Gill, D. S.; Damle, N. K. Curr. Opin. Biotechnol. 2006, 17, 653-658. (4) Minussi, R. C.; Pastore, G. M.; Durán, N. Trends Food Sci. Technol. 2002, 13, 205-216. (5) Medini, K.; Harris, P. W.; Hards, K.; Dingley, A. J.; Cook, G. M.; Brimble, M. A. Chembiochem 2015, 16, 328-336. (6) Singh, P.; Abeysinghe, T.; Kohen, A. Molecules 2015, 20, 1192-1209. (7) Jahangiri, E.; Reichelt, S.; Thomas, I.; Hausmann, K.; Schlosser, D.; Schulze, A. Molecules 2014, 19, 11860-11882. (8) Espinoza-Montero, P. J.; Moreno-Narváez, M. E.; Frontana-Uribe, B. A.; Stojanoff, V.; Moreno, A. Cryst. Growth Des. 2013, 13, 590-598. (9) Dong, C.; Zhang, C. Y.; Liu, Y. Y.; Zhou, R. B.; Cheng, Q. D.; Yin, D. C. Cryst. Growth Des. 2016, 16, 569-577. (10) Lu, Q. Q.; Zhang, B.; Tao, L.; Xu, L.; Chen, D.; Zhu, J.; Yin, D. C. Cryst. Growth Des. 2016, 16, 4869-4876. (11) Schubert, R.; Meyer, A.; Baitan, D.; Dierks, K.; Perbandt, M.; Betzel, C. Cryst. Growth Des. 2017, 17, 954-958. (12) Khurshid, S.; Saridakis, E.; Govada, L.; Chayen, N. E. Nat. Protoc. 2014, 9, 1621-1633. (13) Thakur, A. S.; Robin, G.; Guncar, G.; Saunders, N. F.; Newman, J.; Martin, J. L.; Kobe, B. PloS One 2007, 2, e1091. (14) Kallio, J. M.; Hakulinen, N.; Kallio, J. P.; Niemi, M. H.; Karkkainen, S.; Rouvinen, J. PloS


43


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 44 of 60

One 2009, 4, e4198. (15) Takehara, M.; Ino, K.; Takakusagi, Y.; Oshikane, H.; Nureki, O.; Ebina, T.; Mizukami, F.; Sakaguchi, K. Anal. Biochem. 2008, 373, 322-329. (16) Chayen, N. E.; Saridakis, E.; El-Bahar, R.; Nemirovsky, Y. J. Mol. Biol. 2001, 312, 591-595. (17) Chayen, N. E.; Saridakis, E.; Sear, R. P. Proc. Natl. Acad. Sci. U. S .A. 2006, 103, 597-601. (18) Guo, Y. Z.; Sun, L. H.; Oberthuer, D.; Zhang, C. Y.; Shi, J. Y.; Di, J. L.; Zhang, B. L.; Cao, H. L.; Liu, Y. M.; Li, J.; Wang, Q.; Huang, H. H.; Liu, J.; Schulz, J. M.; Zhang, Q. Y.; Zhao, J. L.; Betzel, C.; He, J. H.; Yin, D. C. Sci. Rep. 2014, 4, 7308. (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. D Biol. Crystallogr. 2005, 61, 53-59. (20) Hemming, S. A.; Bochkarev, A.; Darst, S. A.; Kornberg, R. D.; Ala, P.; Yang, D. S.; Edwards, A. M. J. Mol. Biol. 1995, 246, 308-316. (21) Misquitta, L. V.; Misquitta, Y.; Cherezov, V.; Slattery, O.; Mohan, J. M.; Hart, D.; Zhalnina, M.; Cramer, W. A.; Caffrey, M. Structure 2004, 12, 2113-2124. (22) Fermani, S.; Falini, G.; Minnucci, M.; Ripamonti, A. J. Cryst. Growth 2001, 224, 327-334. (23) Rong, L.; Komatsu, H.; Yoshizaki, I.; Kadowaki, A.; Yoda, S. J. Synchrotron Radiat. 2004, 11, 27-29. (24) Ino, K.; Udagawa, I.; Iwabata, K.; Takakusagi, Y.; Kubota, M.; Kurosaka, K.; Arai, K.; Seki, Y.; Nogawa, M.; Tsunoda, T.; Mizukami, F.; Taguchi, H.; Sakaguchi, K. PloS One 2011, 6, e22582. (25) Gully, B. S.; Zou, J.; Cadby, G.; Passon, D. M.; Iyer, K. S.; Bond, C. S. Nanoscale 2012, 4, 5321-5324.


44

Page 45 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(26) Ghatak, A. S.; Ghatak, A. Langmuir 2013, 29, 4373-4380. (27) McPherson, A.; Shlichta, P. Science 1988, 239, 385-387. (28) Sugahara, M.; Asada, Y.; Morikawa, Y.; Kageyama, Y.; Kunishima, N. Acta Crystallogr. D 2008, 64, 686-695. (29) Sugahara, M.; Kageyama-Morikawa, Y.; Kunishima, N. Cryst. Growth Des. 2011, 11, 110-120. (30) D'Arcy, A.; Bergfors, T.; Cowan-Jacob, S. W.; Marsh, M. Acta Crystallogr. F Struct. Biol. Commun. 2014, 70, 1117-1126. (31) van de Manakker, F.; Vermonden, T.; van Nostrum, C. F.; Hennink, W. E. Biomacromolecules 2009, 10, 3157-3175. (32) Szente, L.; Szejtli, J. Trends Food Sci. Technol. 2004, 15, 137-142. (33) Astray, G.; Gonzalez-Barreiro, C.; Mejuto, J. C.; Rial-Otero, R.; Simal-Gándara, J. Food Hydrocoll. 2009, 23, 1631-1640. (34) Del Valle, E. M. M. Process Biochem. 2004, 39, 1033-1046. (35) Hundre, S. Y.; Karthik, P.; Anandharamakrishnan, C. Food Chem. 2015, 174, 16-24. (36) Mosinger, J.; Tománková, V.; Němcová, I.; Zýka, J. Anal. Lett. 2001, 34, 1979-2004. (37) Juvancz, Z.; Szejtli, J. TrAC Trends Anal. Chem. 2002, 21, 379-388. (38) Shin, J. A.; Lim, Y. G.; Lee, K. H. J. Org. Chem. 2012, 77, 4117-4122. (39) Singh, M.; Sharma, R.; Banerjee, U. C. Biotechnology Adv. 2002, 20, 341-359. (40) Serio, N.; Roque, J.; Badwal, A.; Levine, M. Analyst 2015, 140, 7503-7507. (41) Alsbaiee, A.; Smith, B. J.; Xiao, L.; Ling, Y.; Helbling, D. E.; Dichtel, W. R. Nature 2016, 529, 190-194. (42) Horský, J.; Pitha, J. J. Inclusion Phenom. Mol. Recognit. Chem. 1994, 18, 291-300.


45


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 46 of 60

(43) Brumshtein, B.; Aguilar-Moncayo, M.; Benito, J. M.; Garcia Fernandez, J. M.; Silman, I.; Shaaltiel, Y.; Aviezer, D.; Sussman, J. L.; Futerman, A. H.; Ortiz Mellet, C. Org. Biomol. Chem. 2011, 9, 4160-4167. (44) Anand, U.; Mukherjee, S. Phys. Chem. Chem. Phys. 2013, 15, 9375-9583. (45) Aachmann, F. L.; Otzen, D. E.; Larsen, K. L.; Wimmer, R. Protein Eng. 2003, 16, 905-912. (46) Vandevenne, M.; Gaspard, G.; Belgsir E. M.; Ramnath, M.; Cenatiempo, Y.; Marechal, D.; Dumoulin, M.; Frere, J. M.; Matagne, A.; Galleni, M.; Filee, P. Biochim. Biophys. Acta 2011, 1814, 1146-1153. (47) Prashar, D.; Cui, D.; Bandyopadhyay, D.; Luk, Y. Y. Langmuir 2011, 27, 13091-13096. (48) Signorell, G. A.; Kaufmann, T. C.; Kukulski, W.; Engel, A.; Remigy, H. W. J. Struct. Biol. 2007, 157, 321-328. (49) Machida, S.; Ogawa, S.; Xiaohua, S.; Takaha, T.; Fujii, K.; Hayashi, K. FEBS Lett. 2000, 486, 131-135. (50) Zhang, C. Y.; Wang, Y.; Schubert, R.; Liu, Y.; Wang, M. Y.; Chen, D.; Guo, Y. Z.; Dong, C.; Lu, H. M.; Liu, Y. M.; Wu, Z. Q.; Betzel, C.; Yin, D. C. Cryst. Growth Des. 2016, 16, 705-713. (51) Zhang, C. Y.; Yin, D. C.; Lu, Q. Q.; Guo, Y. Z.; Guo, W. H.; Wang, X. K.; Li, H. S.; Lu, H. M.; Ye, Y. J. Cryst. Growth Des. 2008, 8, 4227-4232. (52) Otwinowski, Z.; Minor, W. Methods Enzymol. 1997, 276, 307-326. (53) Collaborative, C. P. Acta Crystallogr. D 1994, 50, 760. (54) He, Y. F.; Fu, P.; Shen, X. H.; Gao, H. C. Micron 2008, 39, 495-516. (55) Coleman, A. W.; Nicolis, I.; Keller, N.; Dalbiez, J. P. J. Incl. Phenom. Mol. Recognit. Chem. 1992, 13, 139-143.


46

Page 47 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(56) Bonini, M.; Rossi, S.; Karlsson, G.; Almgren, M.; Lo Nostro, P.; Baglioni, P. Langmuir 2006, 22, 1478-1484. (57) Kwon, J. H.; Kim, C. W. J. Cryst. Growth 2004, 263, 536-543.


47


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 48 of 60

For Table of Contents Use Only Utilization of cyclodextrins and its derivative particles as nucleants for protein crystallization Xue-Zhou Yanga, Chen-Yan Zhanga,*, Qian-Jin Wangb, Yun-Zhu Guoa,§, Chen Donga, Er-Kai Yana, Wen-Jing Liua, Xi-Wang Zhenga, Da-Chuan Yina,* a


School of Life Sciences, Northwestern Polytechnical University, Xi’an 710072, Shaanxi, PR China b


Cyclodextrins can be useful as nucleants and they can be explored for use in the mass purification of proteins for the biopharmaceutical industry. Furthermore, this study pointed toward a way to find new nucleants based on the charge of proteins in a solution: the nucleants should preferably be of the opposite charge from the target protein.


48

Page 49 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 1. The structure of β-CD and their derivatives used in this study. (a1), The chemical structure of βCD, (a2), schematic drawing of the cyclodextrin cylinder. (b1) - (b6), the chemical structure of the derivatives PTCD, MHCD, MCD, MECD, MOCD, and HPCD. PCD is polymer of β-CD, the number of monomer is uncertain, so its structure is not shown in Figure 1. 109x113mm (300 x 300 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 2. Normalized crystallization success rate of lysozyme using different amounts of β-CD and its derivatives in the reproducibility study. Two initial concentrations (after mixing) of lysozyme were used: (a) 15 and (b) 20 mg/ml, and the concentration of NaCl was 50 mg/ml. (a) 15 and (b) 20 mg/ml, and the concentration of NaCl was 50 mg/ml. (c) the average normalized crystallization success rate, (d) best normalized crystallization success rate. Error bar was the standard deviation value. Welch’s t-test was applied for the comparison. Two-tailed test was used. The data fit to Gaussian distribution which was tested by K-S test, the variation of two groups was unequal which was tested by F-test, so we used Welch’s t-test for the statistical analysis. 109x93mm (300 x 300 DPI)


Page 50 of 60

Page 51 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 3. Example images of crystallization droplets with different amounts of β-CD and its derivatives. The amount of β-CD, PTCD, PCD, MHCD or MCD in each crystallization droplet was 0 mg/well, 0.002 mg/well, 0.01 mg/well, 0.02 mg/well, 0.1 mg/well and 0.2 mg/well, respectively. Lysozyme and NaCl concentrations were 20 and 50 mg/ml after mixing. It should be noted that the cyclodextrin was easy to be aggregated, even when the concentration of β-CD was 1 mg/ml (0.002 mg/well) (Fig. S1), and these aggregates were difficult to reduce or remove54. 109x107mm (300 x 300 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 4. Comparison of crystallization screening hits with and without adding cyclodextrins. β-CD and its derivatives were used: (a1) and (a2) are β-CD (p = 0.00003 < 0.001); (b1) and (b2) are PTCD (p = 0.0006 < 0.001); (c1) and (c2) are PCD (p = 0.0002 < 0.001); (d1) and (d2) are MHCD (p = 0.00009 < 0.001); (e1) and (e2) are MCD (p = 0.0013 < 0.001); (f1) comparison of the number of unique hits with and without adding cyclodextrins; (f2) comparison of the average normalized unique hits with and without adding cyclodextrins (p = 0.00003 < 0.001). All of the results showed a positive effect, promoting protein crystallization, of all five types of cyclodextrins. Error bar was the standard deviation value. Non-parametric method Kolmogorov-Smirnov test was applied for the statistical analysis, two-tailed test was chosen. 150x142mm (300 x 300 DPI)


Page 52 of 60

Page 53 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 5. Examples of crystallization droplets images with and without the addition of cyclodextrins. The images show droplets of concanavalin in H7 (No. 91 from IndexTM: 0.15 M DL-Malic acid pH 7.0, 20% w/v Polyethylene glycol 3350) of the screening kit IndexTM, glucose isomerase in E6 (No. 54 from IndexTM: 0.05 M Calcium chloride dehydrate, 0.1 M Bis-Tris pH 6.5, 30% v/v Polyethylene glycol monomethyl ether 550), proteinase K in C9 (No. 33 from IndexTM: 1.1 M Sodium malonate pH 7.0, 0.1 M HEPES pH 7.0, 0.5% v/v Jeffamine ® ED-2001 pH 7.0), chymotrypsinogen in G3 (No. 75 from IndexTM: 0.2 M Lithium sulfate monohydrate, 0.1 M Bis-Tris pH 6.5, 25% w/v Polyethylene glycol 3350) and lysozyme in B3 (No. 15 from IndexTM: 0.1 M HEPES pH 7.5, 0.5 M Magnesium formate dihydrate). 109x90mm (300 x 300 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 6. Comparison of the quality of lysozyme crystals grown with and without adding cyclodextrins. (a1) (a2) are the resolution limit of lysozyme crystals grown with and without cyclodextrins (including β-CD, PTCD and MHCD). In (a1), p = 0.045 < 0.05 for MHCD, in (a2), p = 0.032 < 0.05; (b1) - (b2) and (c1) (c2) are the B factor and the mosaicity of lysozyme crystals with and without adding cyclodextrins, respectively. Error bar was the standard deviation value. Two-tailed test was used. The data fit to Gaussian distribution which was tested by K-S test, the variation of two groups was unequal which was tested by Ftest, so we used Welch’s t-test for the statistical analysis. 80x166mm (300 x 300 DPI)


Page 54 of 60

Page 55 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 7. Comparison of the quality of proteinase K crystals grown with and without adding cyclodextrins (MHCD). (a) the resolution limit of proteinase K crystals with and without adding MHCD (p = 0.015 < 0.05); (b) and (c), the B factor and the mosaicity of proteinase K crystals with and without adding MHCD, respectively. Error bar was the standard deviation value. Two-tailed test was used. The data fit to Gaussian distribution which was tested by K-S test, the variation of two groups was equal which was tested by F-test, so we used Student’s t-test for the statistical analysis. 99x52mm (300 x 300 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 8. Particle size measurement results in the solutions of protein, cyclodextrins, and protein with added cyclodextrins. The particle size distributions in the solutions upon adding β-CD, PTCD, PCD, MHCD and MCD are shown in (a), (b), …, (e), respectively. Solution 1 (the red dotted curves): crystallization solution of lysozyme. Concentration of lysozyme was 15 mg/ml, concentration of NaCl was 50 mg/ml. Solution 2 (the green dashed curves): cyclodextrin solution (concentration of β-CD, PCD, and MHCD was 5 mg/ml; for PTCD and MCD: 10 mg/ml; for NaCl: 50 mg/ml). Solution 3 (the purple solid curves): solution with lysozyme and cyclodextrins mixed. 109x121mm (300 x 300 DPI)


Page 56 of 60

Page 57 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 9. Zeta potential measurement results in solutions of protein, of cyclodextrins, and of protein with the addition of cyclodextrins. The concentration of lysozyme was 15 mg/ml; the concentration for β-CD, PCD, and MHCD was 5 mg/ml; the concentration for PTCD and MCD was 10 mg/ml.Error bar was the standard deviation value. t-test was applied for the comparison, two-tailed test was used for the statistical analysis. 109x115mm (300 x 300 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 10. Model of the protein molecules adsorbed onto cyclodextrin molecules during crystallization. 129x36mm (300 x 300 DPI)


Page 58 of 60

Page 59 of 60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 11. The correlation between size increase of particle (black dot) and normalized crystallization success rate (red square) upon adding cyclodextrins. 80x58mm (300 x 300 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 12. Correlation between particle charge and crystallization screening hits. (a) The crystallization screening hits in different groups with cyclodextrins and proteins having the same or opposite charge. N-N: negatively charged cyclodextrin with negatively charged proteins; N-P: negatively charged cyclodextrin with positively charged proteins; P-P: positively charged cyclodextrin with positively charged proteins; P-N: positively charged cyclodextrin with negatively charged proteins. Comparison between control and N-P: p = 0.028 < 0.05. (b) Summarized comparison of crystallization screening hits between cases with cyclodextrins and proteins having the same or opposite charge. Comparison between control and same charge: p = 0.009 < 0.01. Comparison between control and opposite charge: p = 0.0046 < 0.01. Error bar is the standard deviation value of all data in the group. Two-tailed test was used. The data fit to Gaussian distribution which was tested by K-S test, the variation of two groups was unequal which was tested by F-test, so we used Welch’s t-test for the statistical analysis. 109x51mm (300 x 300 DPI)


Page 60 of 60

Utilization of Cyclodextrins and Its Derivative Particles as Nucleants for

Recommend Documents