Co-reporter:Xue-Zhou Yang, Chen-Yan Zhang, Qian-Jin Wang, Yun-Zhu Guo, Chen Dong, Er-Kai Yan, Wen-Jing Liu, Xi-Wang Zheng, and Da-Chuan Yin
Crystal Growth & Design December 6, 2017 Volume 17(Issue 12) pp:6189-6189
Publication Date(Web):October 20, 2017
DOI:10.1021/acs.cgd.7b00455
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 the opposite between the overall charge of target protein and the zeta potential of the cyclodextrin particle.
Co-reporter:Hui-Ling Cao, Li-Hua Sun, Li Liu, Jian Li, Lin Tang, Yun-Zhu Guo, Qi-Bing Mei, Jian-Hua He, Da-Chuan Yin
Journal of Molecular Structure 2017 Volume 1127() pp:1-5
Publication Date(Web):5 January 2017
DOI:10.1016/j.molstruc.2016.07.057
•The paper determined the crystal structures of rHSA at resolution limit of 2.22 Å and HSA at 2.30 Å, which were deposited in PDB with accession codes 4G03 (rHSA) and 4G04 (HSA).•rHSA and HSA achieved 99% structural similarity and rHSA can potentially replace HSA for clinical or other applications.•rHSA can potentially replace HSA. The study provides a theoretical and experimental basis for rHSA applications.Recombinant human serum albumin (rHSA) is potential alternatives for human serum albumin (HSA) which may ease severe shortage of HSA worldwide. In theory, rHSA and HSA are the same. Structure decides function. Therefore, the 3D structural consistency analysis of rHSA and HSA is outmost importance, which is the base of their function consistency. In this paper, the crystal structures of rHSA at resolution limit of 2.22 Å and HSA at 2.30 Å were determined by X-ray diffraction (XRD), which were deposited in the Protein Data Bank (PDB) with accession codes 4G03 (rHSA) and 4G04 (HSA). The differences between rHSA and HSA were systematically analyzed from the crystallization behavior, diffraction data and three-dimensional (3D) structure. The superimposed contrasted analysis indicated that rHSA and HSA achieved a structural similarity of 99% with an r.m.s. deviation of 0.397 Å for the corresponding overall Cα atoms. In addition, the number of α-helices in the rHSA or HSA molecule was verified to be 30. As a result, rHSA can potentially replace HSA. The study provides a theoretical and experimental basis for the clinical and additional applications of rHSA. Meanwhile, it is also a good example for applications of genetic engineering.The figure shows 3D structures of rHSA(A), HSA (B) and superimposed contrast result(C). The superimposed contrasted analysis indicated that rHSA and HSA achieved a structural similarity of 99% with an r.m.s. deviation of 0.397 Å for the corresponding overall Cα atoms. The analysis results indicated that 3D structures of rHSA and HSA were virtually identical and rHSA can potentially replace HSA. The study provides a theoretical and experimental basis for rHSA applications.
Co-reporter:Hai Hou, Bo Wang, Shan-Yang Hu, Meng-Ying Wang, Jinyu Feng, Peng-Peng Xie, Da-Chuan Yin
Journal of Crystal Growth 2017 Volume 468(Volume 468) pp:
Publication Date(Web):15 June 2017
DOI:10.1016/j.jcrysgro.2016.10.007
•The most suitable oxidation condition for screening test was proposed.•There is an optimum treatment process for best protein crystallization trails.•Surface treatment method has practical applicability in crystallization screening.Surface treatment by oxidizing the crystallization plates can significantly promote protein crystallization and requires no change to routine screening protocols; therefore, it is potentially useful for practical protein crystallization screening. However, experiments have shown that the amount of oxidants and the treatment process need to be optimized to achieve effective results. Searching for the suitable amount of oxidants, temperature and processing time for surface treatment of the plate will increase the workload and decrease the efficiency of the screening process. To solve this problem, a series of trials to determine suitable surface treatment conditions were conducted. Based on these experiments, not only was the most suitable processing condition for the optimal protein crystallization screening hits discovered but also the relationship between the treatment process and the protein crystallization screening hits was explored. With these results, the surface treatment of protein crystallization plates became easier and more effective, allowing the oxidizing surface treatment method to be applied on plates for use in routine protein crystallization screening.
Co-reporter:Ren-Bin Zhou;Hui-Ling Cao;Chen-Yan Zhang
CrystEngComm (1999-Present) 2017 vol. 19(Issue 8) pp:1143-1155
Publication Date(Web):2017/02/20
DOI:10.1039/C6CE02562E
The elucidation of protein structures by X-ray crystallography remains the most effectual method to provide accurate structural details at atomic resolution for rational drug design and other biotechnological research studies. Also, emerging applications of protein crystals as ordered nanostructure scaffolds for catalysis, imaging, and drug delivery are attracting much attention. However, the first step of these applications is obtaining high-quality crystals, which is still an obstacle. Successful crystallization requires two steps: nucleation and crystal growth, while the nucleation is a precondition for harvesting the crystal of interest. So controlling protein nucleation may be an alternative breakthrough for this bottleneck. It is well known that nucleants can induce protein crystallization and improve crystal quality, so investigation on the nucleants that can be universally used for any protein crystallization is ongoing. This manuscript reviews the advances that have been achieved using nucleants in protein crystallization and it is a suitable reference for practical crystallization.
Co-reporter:Rui-Qing Chen;Qing-Di Cheng;Jing-Jie Chen;Da-Shan Sun;Liang-Bo Ao;Da-Wei Li;Qin-Qin Lu
CrystEngComm (1999-Present) 2017 vol. 19(Issue 5) pp:860-867
Publication Date(Web):2017/01/30
DOI:10.1039/C6CE02136K
Protein crystallization occurs when the solution conditions are suitable for nucleation and growth of the crystals. pH, as one of the most important parameters that governs the protein crystallization process, can affect the conformation, activity, electrostatic interactions and solubility of protein in the solution. Hence, manipulating the pH is an important path to success in crystallizing proteins. In this paper, we show that varying pH levels over the incubation time during the crystallization process can help increase the chance of obtaining protein crystals. The process of varying the pH can be considered as one way to automatically search for a suitable pH for successful crystallization. This discovery can guide us to develop a new screening strategy in which varying the pH against the incubation time is the major process used during the crystallization screening. Our screening experiments verified that varying the pH is indeed a promising method to achieve efficient crystallization screening of a protein.
Co-reporter:Chen Dong, Xue-Zhou Yang, Chen-Yan Zhang, Yang-Yang Liu, Ren-Bin Zhou, Qing-Di Cheng, Er-Kai Yan, Da-Chuan Yin
Progress in Biophysics and Molecular Biology 2017 Volume 126(Volume 126) pp:
Publication Date(Web):1 July 2017
DOI:10.1016/j.pbiomolbio.2017.02.002
•We review the structural information, biological function, related diseases of MEF2C.•MEF2C plays roles in muscle, neural, chondroid, immune, and endothelial cells.•MEF2C mainly exists as a transcription factor in muscle cells.•MEF2C participates in many physiological events relying on interactions with other proteins.Myocyte enhancer factor 2C (MEF2C) is a transcription factor of MADS box family involved in the early development of several human cells including muscle (i.e., skeletal, cardiac, and smooth), neural, chondroid, immune, and endothelial cells. Dysfunction of MEF2C leads to embryo hypoplasia, disorganized myofibers and perinatal lethality. The main role of MEF2C is its regulation of muscle development. It has been reported that MEF2C-knockout mice die on embryonic day 9.5 from unnatural development of cardiovascular. The effects of MEF2C are mediated by its directly-interacting proteins; therefore, the investigation of these interactions is critical in order to clarify MEF2C's biological function. In this study, we review twenty-five proteins that directly interact with MEF2C, including nineteen proteins related to muscle development, four proteins related to neural cell development, one protein related to chondroid cell development, four proteins related to immune cell development, and two proteins related to endothelial cell development. Among these proteins, the interaction of MEF2C with MRFs is important for differentiation of developing muscle cells. MEF2C interacts with Sox18 for endothelial vessel morphogenesis. The interaction of MEF2C with Cabin1 is important for maintaining T-cell inactivation. Investigating the interactions of MEF2C and its directly-interacting proteins is not only helpful to understand of the physiological function of MEF2C, but also provides a target for future rational drug design.
Co-reporter:Da-Wei Li, Feng-Li He, Jin He, Xudong Deng, Ya-Li Liu, Yang-Yang Liu, Ya-Jing Ye, Da-Chuan Yin
Carbohydrate Polymers 2017 Volume 178(Volume 178) pp:
Publication Date(Web):15 December 2017
DOI:10.1016/j.carbpol.2017.09.035
•The SF/CS scaffold exhibited promising characteristics for 3D cell culture.•Cells in the SF/CS scaffold showed multiple morphologies.•The SF/CS scaffold provided an environment for long-term, sustainable cell culture.•Osteogenic differentiation in the SF/CS scaffold showed different features compared to 2D cases.It has been widely accepted that cell culture in two-dimensional (2D) conditions may not be able to represent growth in three-dimensional (3D) conditions. Systematic comparisons between 2D and 3D cell cultures are needed to appropriately use the existing 2D results. In this work, we conducted a comparative study between 2D and 3D cell cultures of MC3T3-E1 using the same type of material (a mixture of silk fibroin (SF) and chitosan (CS)). Our results showed 3D SF/CS scaffold exhibited different effects on cell culture compared with the 2D cases. 1) The cells grown in 3D scaffold showed multiple morphologies. 2) The proliferation of cells in 3D scaffold was long-term and sustainable. 3) Cell differentiation occurred throughout the entire 3D scaffold. The results showed that cell culture in 3D SF/CS scaffold exhibited different features than 2D cases and 3D SF/CS scaffold could be a promising material for 3D cell culture.
Co-reporter:Hai Hou;Bo Wang;Peng-Peng Xie;Yun-Zhu Guo;Jin Li
CrystEngComm (1999-Present) 2017 vol. 19(Issue 24) pp:3237-3243
Publication Date(Web):2017/06/20
DOI:10.1039/C7CE00664K
We presented a systematic method that combined the cross-diffusion microbatch method (CDM) with the seeding technique to enhance protein crystal reproducibility based on a “common dispersing agent”. The common dispersing agent is used to dilute the crystal seeds. Seven proteins were screened, and more crystallization conditions were found. X-ray diffraction showed that the combined method is practical and useful for obtaining high-quality protein crystals.
Co-reporter:Qin-Qin Lu, Bin Zhang, Liang Tao, Lu Xu, Da Chen, Jing Zhu, and Da-Chuan Yin
Crystal Growth & Design 2016 Volume 16(Issue 9) pp:4869
Publication Date(Web):July 18, 2016
DOI:10.1021/acs.cgd.6b00227
Growing high quality protein crystals enables higher quality high-resolution structure determination by X-ray diffraction. In this paper, we show that mechanical vibration could be an effective and useful tool for improving protein crystal quality. The crystal quality of the selected proteins was examined using X-ray diffraction. The quality comparison of the crystals grown with and without vibration verified that mechanical vibration not only enhanced the morphology of the crystals but also improved the crystal quality. Therefore, we propose that mechanical vibration could be a useful alternative tool for obtaining high quality-diffracting protein crystals.
Co-reporter:Yong-Ming Liu, Hai-Sheng Li, Zi-Qing Wu, Rui-Qing Chen, Qin-Qin Lu, Yun-Zhu Guo, Chen-Yan Zhang and Da-Chuan Yin
CrystEngComm 2016 vol. 18(Issue 9) pp:1609-1617
Publication Date(Web):25 Jan 2016
DOI:10.1039/C6CE00060F
Environments with varying temperatures have been shown to beneficially increase the probability of obtaining protein crystals. Therefore, a cycling temperature strategy (CTS) has been proposed for protein crystallization screening. During the practical application of this strategy, it is necessary to know the effective temperature range that promotes crystallization to design a suitable temperature program. In this paper, the effects of different temperature ranges on lysozyme crystallization (or more specifically, nucleation) were investigated. The results show that a small periodic variation in the temperature range of as little as 0.4 K can have a significant effect on the crystallization success rate under some crystallization concentration conditions, confirming that crystallization of lysozyme is very sensitive to temperature variation. Because practical protein crystallization is always performed in an environment with slight temperature variations, the sensitivity of protein crystallization to temperature may provide an explanation for the poor reproducibility of protein crystallization. Further investigation of the CTS on lysozyme crystallization showed that a cycling temperature strategy exerts an effect on protein crystallization by altering the supersaturation caused by changes in temperature.
Co-reporter:Chen Dong, Chen-Yan Zhang, Yang-Yang Liu, Ren-Bin Zhou, Qing-Di Cheng, and Da-Chuan Yin
Crystal Growth & Design 2016 Volume 16(Issue 2) pp:569-577
Publication Date(Web):January 6, 2016
DOI:10.1021/acs.cgd.5b00702
Protein crystallizes at specific conditions (correct precipitants, appropriate concentrations of protein and precipitants, suitable pH and correct temperature, etc.). If the conditions are not appropriate, crystallization will not occur. In protein crystallization screening, the target protein is mixed one by one with many chemical agents and then incubated at a set temperature. If the concentrations of the chemical agents and the target proteins are not in a range suitable for crystallization, the crystallization will not occur. To expand the concentration screening space, we propose in this paper a new design of protein crystallization plates for cross-diffusion microbatch and microbatch methods. The new plates have 96 units corresponding to the conditions of the commercial screening kits, and each unit contains four wells for holding the crystallization droplets. By dispensing crystallization droplets to the four wells at different volume ratios of protein to precipitant solutions, we can obtain four different initial concentrations for each unit, and thus a wider concentration range can be screened. The comparison between the screening performance of the new plates and the traditional sitting-drop vapor diffusion plate showed that the new design of the plates exhibited significantly improved results in obtaining more crystallization conditions.
Co-reporter:Da-Chuan Yin
Progress in Crystal Growth and Characterization of Materials 2015 Volume 61(Issue 1) pp:1-26
Publication Date(Web):March 2015
DOI:10.1016/j.pcrysgrow.2015.03.001
The rapid advance in superconducting magnet technology enables more and more applications for the use of high magnetic fields in scientific researches and industrial manufacturing. These applications include material processing, separation, chemical reaction, nuclear fusion, high energy physics, and many more. Generally, a superconducting magnet provides both homogeneous and inhomogeneous magnetic fields simultaneously, and both can affect the samples in the field so that the magnetic field can be utilized for various purposes. A homogeneous or inhomogeneous magnetic field will exert a torque on suspending particles in a solution if the particles have anisotropic magnetic susceptibility, which will further influence the properties of the solution; in an inhomogeneous magnetic field, a repulsive force will act on a diamagnetic solution so that the levels of apparent or effective gravity of the solution can be tuned in a vertical magnetic field. These effects can be utilized to govern the physical and chemical processes in solution like crystallization. In recent years, high magnetic fields have been applied in protein crystallization. It was found that a magnetic field can align the crystals along the field direction, decrease the diffusivity of macromolecules in the solution, and increase the viscosity of the solution; a suitable inhomogeneous magnetic field can damp the natural convection substantially, which resembles the case in a space environment. Both homogeneous and inhomogeneous magnetic fields have been found to improve the quality of some protein crystals. These discoveries showed that the researches on protein crystallization in high magnetic field is potentially valuable, because obtaining high quality protein crystals is important for 3-dimensional structure determination of proteins using X ray crystallography. This paper will review the background and more recent progress and discuss the future perspectives in this research field.
Co-reporter:Lin-Jun Huang, Hui-Ling Cao, Ya-Jing Ye, Yong-Ming Liu, Chen-Yan Zhang, Qin-Qin Lu, Hai Hou, Peng Shang and Da-Chuan Yin
CrystEngComm 2015 vol. 17(Issue 6) pp:1237-1241
Publication Date(Web):13 Nov 2014
DOI:10.1039/C4CE01892C
We present a new method for the realization of high-throughput protein crystallization screening using an array of 96 capillaries aligned in a circle. In this method, each capillary represents a single crystallization condition, and all capillaries experience identical magnetic field conditions. After crystallization, the crystals in the capillary can be directly diffracted without harvesting. This method proved easy to perform and is applicable for use in magnetic fields and may be further extended for use in other circumstances, for example, under space microgravity conditions.
Co-reporter:Yue Liu, Xian-Fang Zhang, Chen-Yan Zhang, Yun-Zhu Guo, Si-Xiao Xie, Ren-Bin Zhou, Qing-Di Cheng, Er-Kai Yan, Ya-Li Liu, Xiao-Li Lu, Qin-Qin Lu, Hui-Meng Lu, Ya-Jing Ye and Da-Chuan Yin
CrystEngComm 2015 vol. 17(Issue 29) pp:5488-5495
Publication Date(Web):05 Jun 2015
DOI:10.1039/C5CE00779H
Crystallisation of proteins is usually achieved with the help of chemical agents. Because there are few general guidelines in determining what agents will help to crystallise a specific protein, suitable crystallisation agents are often found via exhaustive trial-and-error tests by mixing many chemical agents (the collection of which is called a crystallisation screening kit) one-by-one with the protein. Currently, many commercially available crystallisation screening kits have been developed and utilised in practical crystallisation screen experiments. However, information regarding the design of new screening kits has yet to be expanded using a large amount of experimental data. Here, we show the step-by-step design processes of a polyethylene glycol-based screening kit. It was found that the screening performance could be improved by modifying the crystallisation screening kits according to the accumulated data (such as those in the Biological Macromolecule Crystallisation Database (BMCD)), the screening test results and existing knowledge. The screening kit designed in this paper can be used for practical protein crystallisation screen experiments and the method can be used in the design of other crystallisation screening kits.
Co-reporter:Hai Hou, Bo Wang, Shan-Yang Hu, Jing-Zhang Wang, Peng-Fei Zhu, Yue Liu, Meng-Ying Wang and Da-Chuan Yin
CrystEngComm 2015 vol. 17(Issue 29) pp:5365-5371
Publication Date(Web):22 Jun 2015
DOI:10.1039/C5CE00551E
We presented a systematic quality comparison of protein crystals grown using the cross-diffusion microbatch (CDM) and standard sitting-drop vapor diffusion methods. Eleven proteins were screened and it was found that crystals grown using CDM exhibited a better morphology. X-ray diffraction showed that the CDM method is practical and useful for obtaining high-quality protein crystals.
Co-reporter:Ya-Jing Ye, Yang-Yang Liu, Da-Chuan Yin
Applied Surface Science 2015 Volume 351() pp:594-599
Publication Date(Web):1 October 2015
DOI:10.1016/j.apsusc.2015.05.181
Highlights
- •
Inducing ability of self-assembly monolayers in large gradient magnetic fields.
- •
Bonding information of functional groups obtained by first principles calculations.
- •
The gravity fields affected the compositions of the apatite layers.
Co-reporter:Er-Kai Yan, Hui-Ling Cao, Chen-Yan Zhang, Qin-Qin Lu, Ya-Jing Ye, Jin He, Lin-Jun Huang and Da-Chuan Yin
RSC Advances 2015 vol. 5(Issue 33) pp:26163-26174
Publication Date(Web):06 Mar 2015
DOI:10.1039/C5RA01722J
Cross-linked protein crystal technology, as either a protein stabilisation or enzyme immobilisation method, has garnered more attention recently. This method not only can retain the original activity of the protein molecule but can also significantly enhance the crystals' mechanical and chemical stability. This review presents the preparation and mechanism of cross-linked protein crystals using glutaraldehyde. The mechanical, chemical and thermal properties of the cross-linked protein crystals are also reviewed in detail. In addition, this paper summarises the applications of cross-linked protein crystals in the fields of materials science, biosensors, chromatographic analysis, oral delivery and protein crystal quality improvement. Finally, the limitations and perspectives on cross-linked protein crystals are presented.
Co-reporter:Qin-Qin Lu;Si-Xiao Xie;Yong-Ming Liu ;Rui-Qing Chen
Crystal Research and Technology 2011 Volume 46( Issue 9) pp:917-925
Publication Date(Web):
DOI:10.1002/crat.201100178
Abstract
In this study, effects of diluting either protein or crystallization agents in the droplets on the success rate of protein crystallization was investigated. Diluting the crystallization agent was found to increase the success rate of protein crystallization. Theoretical analysis showed that, concentration ranges of both protein and crystallization agent that can be scanned during the vapor diffusion process are wider with diluting the crystallization agent than that without dilution, resulting in more opportunities for the crystallization solution to be in the nucleation zone. On the other hand, diluting protein could lead to controversial results depending on the location of the initial concentration relative to that of the nucleation zone in the phase diagram. The method of diluting the crystallization agent is therefore proposed as an alternative modification to the conventional vapor diffusion method for obtaining more crystallization conditions in protein crystallization screening. (© 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)
Co-reporter:Yun-Zhu Guo, ;Qin-Qin Lu;Xi-Kai Wang ;Jun Liu
Crystal Research and Technology 2010 Volume 45( Issue 2) pp:158-166
Publication Date(Web):
DOI:10.1002/crat.200900609
Abstract
We examined a simple approach, i.e., etching cover glasses using hydrofluoric acid (HF), to determine whether cover glass treatment enhances nucleation in hanging drop protein crystallization. Hen egg white lysozyme and proteinase K were used as the model proteins. We found that the treatment increased the success rate of crystallization. The results indicated that the simple treatment, which is easy to adopt without changing much in the hanging drop method, can be utilized as an alternative method to enhance protein crystallization screens (© 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)
Co-reporter:Xi-Kai Wang, ;Chen-Yan Zhang;Qin-Qin Lu;Yun-Zhu Guo ;Wei-Hong Guo
Crystal Research and Technology 2010 Volume 45( Issue 5) pp:479-489
Publication Date(Web):
DOI:10.1002/crat.201000097
Abstract
Varying the temperature has been proven to be beneficial for improving the screening efficiency of protein crystallisation, and thus a crystallisation screening strategy based on this phenomenon can be developed. Such a temperature varying strategy can be applied in practical crystallisation screening, however, there are no guidelines for determining what temperature programme should be utilised. It is therefore necessary to investigate how the temperature programme affects the crystallisation process, so as to help people design a suitable temperature programme. For this purpose, we investigated the effect of temperature programmes on the protein crystallisation (lysozyme, proteinase K, and concanavalin A) that are characterised by different solubility behaviours with respect to temperature. Judging from the reproducibility studies of protein crystallisation with different temperature programmes, we recommend using linear temperature programmes for a moderate time period (24 to 48 h) and a large temperature range according to the properties of the proteins. (© 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)
Co-reporter:Da-Chuan Yin, Li-Qiang Geng, Qin-Qin Lu, Hui-Meng Lu, Peng Shang and Nobuko I. Wakayama
Crystal Growth & Design 2009 Volume 9(Issue 12) pp:5083-5091
Publication Date(Web):September 23, 2009
DOI:10.1021/cg900202e
We investigated the effect of paramagnetic salts (NiCl2, CoCl2, and MnCl2) on the magnetic orientation of hen-egg white lysozyme (HEWL) crystals at different pHs. Multiple orientation responses of the crystals to magnetic fields were discovered when NiCl2 and CoCl2 were used as the crystallization agents (the c-axis and the ⟨110⟩ orientations for NiCl2 solution, and the c-axis, the a-axis and the ⟨110⟩ orientations for CoCl2 solution). Only the normal c-axis orientation was found when MnCl2 was used as the crystallization agent. The multiple orientation responses were sensitive to the variations in the pH. These phenomena are discussed in the context of the incorporation of paramagnetic cations into lysozyme. Although anions affect the crystallization of lysozyme, the effect of cations on the crystallization of lysozyme has been considered marginal. This study reveals that cations can also exhibit profound effects on the crystallization of lysozyme.
Co-reporter:Chen-Yan Zhang, Da-Chuan Yin, Qin-Qin Lu, Yun-Zhu Guo, Wei-Hong Guo, Xi-Kai Wang, Hai-Sheng Li, Hui-Meng Lu and Ya-Jing Ye
Crystal Growth & Design 2008 Volume 8(Issue 12) pp:4227-4232
Publication Date(Web):October 22, 2008
DOI:10.1021/cg800689j
Temperature is generally considered as an important factor in protein crystallization. Such is true because crystals usually grow at a preferable temperature in a certain crystallization solution. If a nonsuitable temperature is used, the solution will not yield crystals. However, it is difficult to decide the best temperature suited for screening the crystallization condition of proteins. In this study, it was found out that, compared to constant temperature, a variation in a reasonable range can result in a more efficient crystallization screening. Using the Sparse Matrix Screen with the screening kit Index, this study tested nine commercially available proteins and proved that, compared to the conventional constant temperature strategy, a varying temperature strategy can actually increase the possibility of obtaining crystals. Consequently, the cycling temperature strategy (CTS) is then proposed to be utilized in most screening tasks when the suitable crystallization temperature is unknown.
Co-reporter:Da-Chuan Yin;Nobuko I. Wakayama;Hui-Meng Lu;Ya-Jing Ye;Hai-Sheng Li;Hui-Min Luo;Yuko Inatomi
Crystal Research and Technology 2008 Volume 43( Issue 4) pp:447-454
Publication Date(Web):
DOI:10.1002/crat.200710998
Abstract
The reproducibility of biomacromolecular crystallization (tetragonal and orthorhombic lysozyme crystals) was studied by monitoring the evolution of protein concentration during the crystallization process using Mach-Zehnder interferometer. It was found that formation of both tetragonal and orthorhombic crystals exhibited poor reproducibility. When the crystallization occurred under isothermal conditions, the protein concentration in the solution varied differently in different experiments under identical conditions (for both types of crystals). Moreover, in the case of orthorhombic lysozyme crystallization (under either isothermal or thermal gradient conditions), it is clear that the crystals could not be always readily formed. When formation of tetragonal lysozyme crystals was conducted at a temperature gradient condition, however, the evolution of concentration was reproducible. The phenomena found in this study revealed that biomacromolecular crystallization can be uncertain, which is probably caused by the process of nucleation. Such uncertainties will be harmful for the efforts of screening crystallization conditions for biomacromolecules. (© 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)
Co-reporter:Ting Huyan, Qi Li, Lin-Jie Ye, Hui Yang, Xiao-Ping Xue, Ming-Jie Zhang, Qing-Sheng Huang, Da-Chuan Yin, Peng Shang
International Immunopharmacology (December 2014) Volume 23(Issue 2) pp:452-459
Publication Date(Web):1 December 2014
DOI:10.1016/j.intimp.2014.09.018
•Tumor cells over-express and secrete human aspartyl β-hydroxylase (HAAH).•HAAH inhibits the cytotoxicity activities of human NK cells.•HAAH induces NK cells apoptosis via caspase signaling pathways.•NKG2D, NKp44 and cytokines of NK cells were down-regulated by HAAH.•HAAH mAbs restore the function of NK cells and promote the ADCC activity of NK cells.Natural killer (NK) cells are a key component of the innate immune system and play pivotal roles as inflammatory regulators and in tumor surveillance. Human aspartyl β-hydroxylase (HAAH) is a plasma membrane and endoplasmic reticulum protein with hydroxylation activity, which is over-expressed in many malignant neoplasms and can be detected from the sera of tumor patients. HAAH is involved in regulating tumor cell infiltration and metastasis. Escaping from immune surveillance may help tumor cell infiltration and metastasis. However, the effects of HAAH on tumor immune surveillance have not yet been investigated carefully. The present study investigated the potential use of HAAH as an immune regulator of human NK cells. We assessed the effects of recombinant HAAH (r-HAAH) on primary human NK cell morphology, viability, cytotoxicity, apoptosis, receptors expression and cytokine/cytolytic proteins production. Our results demonstrated that r-HAAH negatively affects NK cell activity in a time and dose-dependent manner. It noticeably reduces the viability of the NK cells by increasing apoptosis and necrosis via caspase signaling pathways. Moreover, r-HAAH reduces the NK cell cytotoxicity by inhibiting surface expression of NKG2D, NKp44 and IFN-γ secretion. These findings suggest that one of the ways by which HAAH actively promotes tumor formation and proliferation is by inhibiting NK cell-surveillance activity.
Co-reporter:Chen-Yan Zhang, Chen Dong, Yue Liu, Bin-Bin Jiang, Meng-Ying Wang, Hui-Ling Cao, Wei-Hong Guo, Da-Chuan Yin
Journal of Crystal Growth (15 May 2015) Volume 418() pp:45-51
Publication Date(Web):15 May 2015
DOI:10.1016/j.jcrysgro.2015.01.035
•The effect of solvent evaporation rate on lysozyme crystallization was studied.•The rate of reaching supersaturation are important for crystallization.•Manipulation of solvent evaporation rate can help to obtain crystals.One well-known prerequisite for successful crystallization from solution is a supersaturated solution. To achieve supersaturation, many methods are known, among which solvent evaporation is a common approach. For protein crystallization, the most widely used method is vapor diffusion, in which solvent evaporation from the crystallization solution is the major reason for achieving supersaturation. The solvent evaporation rate may affect the actual concentration distribution in the crystallization solution, thereby influencing the crystallization process. To explore the effect of evaporation rate on protein crystallization, we used lysozyme as a model protein and studied the crystallization success rate at different evaporation conditions. Successful crystallization occurred only when both supersaturation and evaporation rates were in suitable ranges. This study demonstrates that both supersaturation level and the rate of reaching supersaturation (or solvent evaporation rate) are important for lysozyme crystallization. To increase the chance of obtaining crystals, manipulation of solvent evaporation rate is one choice. According to this assumption, we performed crystallization screening trials at different evaporation rates using three model proteins. The trials demonstrate that control of the evaporation rate during crystallization may provide more opportunities to obtain crystals.
Co-reporter:Chen-Yan Zhang, Chen Dong, Xiao-Li Lu, Bei Wang, Tian-Yuan He, Rui-Zeng Yang, Hua-Long Lin, Xue-Zhou Yang, Da-Chuan Yin
Journal of Crystal Growth (1 April 2017) Volume 463() pp:72-78
Publication Date(Web):1 April 2017
DOI:10.1016/j.jcrysgro.2017.01.036
•A simpler and faster pH buffer strategy is proposed.•pH-controlling buffer is added to crystallization droplet to adjust its pH.•Protein crystallization is enhanced due to expansion of pH space.We have proposed a rational strategy for selecting a suitable pH of protein solution based on protein biochemical properties. However, it is difficult to use this strategy for biochemical properties unknown proteins. In this paper, a simpler and faster pH buffer strategy was proposed. An additional pH-controlling buffer was added to crystallization droplet mixed with protein solution and commercial crystallization reagents to adjust its pH. The results revealed that protein crystallization success rates were enhanced by this strategy due to expansion of the pH screening space, which was closely related with protein solubility. Thus, the possibility of reaching supersaturation was increased by using this strategy.
