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生物大分子(如蛋白质等)在生物体内行使功能必须要保证其立体结构的正确性。作为研究大分子高级结构的主要手段,结晶技术结合X-射线衍射技术、核磁共振技术以及电镜技术被普遍应用于高级结构的数据分析。随着这些技术的进一步完善,目前已经能完成蛋白质与配体的共结晶。遗传信息从最原始的DNA或RNA传递到以蛋白质的形式呈现功能的过程是由众多酶或蛋白质复合体催化的多步骤进程,解析其中重要元件的空间结构推动了对这些酶反应机理的深入研究。主要阐述结晶技术在遗传信息传递过程中关键酶或蛋白质复合体的研究中的应用。 相似文献
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NaCl溶液体系溶菌酶结晶相图的测定 总被引:1,自引:0,他引:1
溶液相图测定是研究蛋白质晶体生长的一重要方法。使用微量配液(Microbatch)自动化结晶技术,对鸡蛋清溶菌酶-NaCl溶液体系的相图进行了较精细的测定,获得该体系的精细相图。该结果反映了蛋白质结晶相图的一般分布规律,并揭示了一些细节特点。实验还表明,微量配液法自动化结晶技术便于这类相图测定,而后者又可提供优化蛋白质结晶条件的有用信息。 相似文献
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蛋白质大分子的结晶 总被引:5,自引:0,他引:5
近几年研究蛋白质晶体的生长机理及过程日益受到重视 ,并逐步发展为蛋白质晶体学中的另一个重要的科学分支。1 .蛋白质结晶的基本过程蛋白质属于生物大分子 ,但其结晶的一般过程与盐、有机小分子等的结晶过程一样可分为 3个阶段进行 :( 1 )形成稳定的晶核 ;( 2 )由晶核生长为晶体 ;( 3)生长结束。1 .1 成核 是指溶解在溶液中的分子或非晶态的低聚体 (二聚体 ,三聚体等 )以过饱和度为驱动力聚合在一起形成热力学稳态的点阵结构。Drenth等[1] 研究发现 ,在蛋白质结晶后其溶液体系的自由能降低了 1 2~ 2 4kJ/mol。另外 ,结晶蛋… 相似文献
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应用Thermal Shift Assays技术,高通量优化了重组森林脑炎病毒丝氨酸蛋白酶纯化过程中所使用的缓冲液中盐的种类和pH,显著改善了该重组蛋白在纯化过程中出现可溶性聚集体的现象。最终获得的重组蛋白质单体量由原先的75%提高到99%以上,极大提高了该纯化蛋白质的均一性和质量,为后续的蛋白质结晶研究奠定了基础。 相似文献
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《核酸和蛋白质结晶》(Crystallization of nucleic acid and proteins)由A.Ducruix和R.Giege编著,1992年IRL出版社出版,331页。最近十年,结晶学成为了解生物大分子结构的关键工具,但是,结晶技术操作起来比较困难。该书对蛋白质和核酸产生结晶的衍射研究提供了详细和合理的指导,并且给予标准结晶方法以及制备和处理大分子的程序。其论题包括:晶种程序;在微重力和超重力下凝胶结晶;核酸,核酸—蛋白质络合物和 相似文献
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综述了杂质对蛋白质晶体生长影响研究领域的进展情况. 对可能的杂质来源以及杂质对结晶过程的影响进行了介绍.重点介绍了和结晶蛋白质分子结构相似的杂质分子的影响, 包括晶体成核、生长形态、表面形貌、生长动力学、质量等,以及杂质在晶体中的重新分配. 相似文献
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蛋白质晶体生长是用衍射法测定和研究蛋白质三维结构不可缺少的首要步骤,因而对于从分子水平了解生命过程和有效地开发蛋白质工程、理性药物设计等新的生物技术具有重要意义。这一结构测定步骤所处的落后状态,更使蛋白质晶体生长成为倍受重视的研究课题。蛋白质和核酸等生物大分子的结晶是一个受多个因素影响的过程。来自不同学科的研究人员从各个方面对蛋白质的晶体生长开展了研究,并取得了不同程度的进展。 相似文献
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S. Schmidt D. Havekost K. Kaiser J. Kauling H.‐J. Henzler 《Engineering in Life Science》2005,5(3):273-276
Protein crystallization offers great potential in downstream processing of pharmaceutical protein active ingredients. The advantages, which are well known and widely utilized in low‐molecular weight crystallization, can also be expected to be found to some extent in protein crystallization. However, there is still a marked need for improvement in two main areas of protein processing, namely, in crystallization from impure solutions and scale‐up. 相似文献
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HUANG Ju-Fu DONG Zhi-Gang WANG Dao-Yong L Yu-Bing ZHANG Hua-Feng WANG Yao-Ping HAN Yi DAI Xiao-Hu 《植物学报(英文版)》2001,43(4):375-379
Under a suitable condition of crystallization, dark brown short rhombohedron crystals could be obtained from nitrogenase MnFe protein purified from a mutant UW3 of Azotobacter vinelandii Lipmann grown in Mn-containing but Mo- and NH3-free medium. The possibility of crystallization, and number,size and quality of crystals were obviously dependent on concentrations of NaCl,MgCl2, PEG 8000,Tris and Hepes buffer and on methods for crystallization. PEG concentration affected on the shape of the crystals.The optimal concentrations of the chemicals for crystallization of MnFe protein were slightly different from those for crystallization of ΔnifZ MoFe protein from a nifZ deleted strain of Azotobacter vinelandii . SDS-PAGE showed that the protein from the dissolved crystals was almost the same as MnFe protein before crystallization, indicating that the crystal was formed from MnFe protein. 相似文献
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Polymer-driven crystallization 总被引:1,自引:0,他引:1
Nauli S Farr S Lee YJ Kim HY Faham S Bowie JU 《Protein science : a publication of the Protein Society》2007,16(11):2542-2551
Obtaining well-diffracting crystals of macromolecules remains a significant barrier to structure determination. Here we propose and test a new approach to crystallization, in which the crystallization target is fused to a polymerizing protein module, so that polymer formation drives crystallization of the target. We test the approach using a polymerization module called 2TEL, which consists of two tandem sterile alpha motif (SAM) domains from the protein translocation Ets leukemia (TEL). The 2TEL module is engineered to polymerize as the pH is lowered, which allows the subtle modulation of polymerization needed for crystal formation. We show that the 2TEL module can drive the crystallization of 11 soluble proteins, including three that resisted prior crystallization attempts. In addition, the 2TEL module crystallizes in the presence of various detergents, suggesting that it might facilitate membrane protein crystallization. The crystal structures of two fusion proteins show that the TELSAM polymer is responsible for the majority of contacts in the crystal lattice. The results suggest that biological polymers could be designed as crystallization modules. 相似文献
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Crystallization has been a bottleneck in the X-ray crystallography of proteins. Although many techniques have been developed to overcome this obstacle, the impurities caused by chemical reactions during crystallization have not been sufficiently considered. Oxidation of proteins, which can lead to poor reproducibility of the crystallization, is a prominent example. Protein oxidization in the crystallization droplet causes inter-molecular disulfide bridge formation, formation of oxidation film, and precipitation of proteins. These changes by oxidation are typically irreversible. The best approach for preventing protein oxidization during crystallization is anaerobic crystallization. Here we review the anaerobic crystallization of proteins, which was originally developed to trap a reaction intermediate of the enzyme in the crystal. We also summarize representative anaerobic crystallizations from our laboratory and the general setup of anaerobic crystallization. 相似文献
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Of many factors affecting protein crystallization, randomness in proteins has been given less attention although highly structured proteins would be at low entropy state. The factors, which impact on protein crystallization, are almost exclusively related to non-random amino acid properties such as physiochemical properties of amino acids. In this study, we used logistic regression and neural network to model the success rate of crystallization of 420 proteins from Staphylococcus aureus with each of non-random and random amino acid properties in order to determine whether randomness in a protein plays a role in the crystallization process. The results show that randomness is indeed involved in the crystallization process, and this rationale would enrich our knowledge on crystallization process and enhance our ability to crystallize more important proteins. 相似文献
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Can the propensity of protein crystallization be increased by using systematic screening with metals?
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Raghurama P. Hegde Gowribidanur C. Pavithra Debayan Dey Steven C. Almo S. Ramakumar Udupi A. Ramagopal 《Protein science : a publication of the Protein Society》2017,26(9):1704-1713
Protein crystallization is one of the major bottlenecks in protein structure elucidation with new strategies being constantly developed to improve the chances of crystallization. Generally, well‐ordered epitopes possessing complementary surface and capable of producing stable inter‐protein interactions generate a regular three‐dimensional arrangement of protein molecules which eventually results in a crystal lattice. Metals, when used for crystallization, with their various coordination numbers and geometries, can generate such epitopes mediating protein oligomerization and/or establish crystal contacts. Some examples of metal‐mediated oligomerization and crystallization together with our experience on metal‐mediated crystallization of a putative rRNA methyltransferase from Sinorhizobium meliloti are presented. Analysis of crystal structures from protein data bank (PDB) using a non‐redundant data set with a 90% identity cutoff, reveals that around 67% of proteins contain at least one metal ion, with ~14% containing combination of metal ions. Interestingly, metal containing conditions in most commercially available and popular crystallization kits generally contain only a single metal ion, with combinations of metals only in a very few conditions. Based on the results presented in this review, it appears that the crystallization screens need expansion with systematic screening of metal ions that could be crucial for stabilizing the protein structure or for establishing crystal contact and thereby aiding protein crystallization. 相似文献
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Myelin basic protein (MBP) is the predominant extrinsic protein in both central and peripheral nervous system myelins. It is thought to be involved in the stabilizing interactions between myelin membranes, and it may play an important role in demyelinating diseases such as multiple sclerosis. In spite of the fact that this abundant protein has been known for almost three decades, its three-dimensional crystal structure has not yet been determined. In this study we report on our extensive attempts to crystallize the major 18.5 kDa isoform of MBP. We used MBP having different degrees of purity, ranging from crude MBP (that was acid or salt extracted from isolated myelin), to highest purity single isoform. We used conventional strategies in our search for a suitable composition or a crystallization medium. We applied both full and incomplete factorial searches for crystallization conditions. We analyzed the available data on proteins which have previously resisted crystallization, and applied this information to our own experiments. Nevertheless, despite our efforts which included 4600 different conditions, we were unable to induce crystallization of MBP. Previous work on MBP indicates that when it is removed from its native environment in the myelin membrane and put in crystallization media, the protein adopts a random coil conformation and persists as a population of structurally non-identical molecules. This thermodynamically preferred state presumably hinders crystallization, because the most fundamental factor of protein crystallization-homogeneity of tertiary structure-is lacking. We conclude that as long as its random coil flexibility is not suppressed, 18.5 kDa MBP and possibly also its isoforms will remain preeminent examples of proteins that cannot be crystallized. 相似文献
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While bulk crystallization from impure solutions is used industrially as a purification step for a wide variety of materials, it is a technique that has rarely been used for proteins. Proteins have a reputation for being difficult to crystallize and high purity of the initial crystallization solution is considered paramount for success in the crystallization. Although little is written on the purifying capability of protein crystallization or of the effect of impurities on the various aspects of the crystallization process, recent published reports show that crystallization shows promise and feasibility as a purification technique for proteins. To further examine the issue of purity in macromolecule crystallization, this study investigates the effect of the protein impurities, avidin, ovalbumin, and conalbumin at concentrations up to 50%, on the solubility, crystal face growth rates, and crystal purity of the protein lysozyme. Solubility was measured in batch experiments while a computer controlled video microscope system was used to measure the ?110? and ?101? lysozyme crystal face growth rates. While little effect was observed on solubility and high crystal purity was obtained (>99.99%), the effect of the impurities on the face growth rates varied from no effect to a significant face specific effect leading to growth cessation, a phenomenon that is frequently observed in protein crystal growth. The results shed interesting light on the effect of protein impurities on protein crystal growth and strengthen the feasibility of using crystallization as a unit operation for protein purification. 相似文献
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Detergents destabilize the cubic phase of monoolein: implications for membrane protein crystallization
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The in meso method for membrane protein crystallization uses a lipidic cubic phase as the hosting medium. The cubic phase provides a lipid bilayer into which the protein presumably reconstitutes and from which protein crystals nucleate and grow. The solutions used to spontaneously form the protein-enriched cubic phase often contain significant amounts of detergents that were employed initially to purify and to solubilize the membrane protein. By virtue of their surface activity, detergents have the potential to impact on the phase properties of the in meso system and, by extension, the outcome of the crystallization process. The purpose of this study was to quantify the effects that a popular series of nonionic detergents, the n-alkyl-beta-D-glucopyranosides, have on the phase behavior of hydrated monoolein, the lipid upon which the in meso method is based. Phase identity and phase microstructure were characterized by small-angle x-ray diffraction on samples prepared to mimic in meso crystallization conditions. Measurements were made in the 0-40 degrees C range. Samples prepared in the cooling direction allow for the expression of metastability, a feature of liquid crystalline phases that might be exploited in low-temperature crystallization. The results show that the cubic phase is relatively insensitive to small amounts of alkyl glucosides. However, at higher levels the detergents trigger a transition to the lamellar phase in a temperature- and salt concentration-dependent manner. These effects have important implications for in meso crystallization. A diffraction-based method for assaying detergents is presented. 相似文献