用于蛋白质结晶的分子改造技术

获得晶体及提高晶体质量是蛋白质结晶方法学中的两大基本问题.为解决这两个问题,结构生物学家已发展了许多方法,其中针对蛋白质本身进行分子改造是非常重要的方法之一.通过蛋白质工程技术,如定点突变、还原甲基化修饰、剪切或删除构象柔性环区、融合蛋白、复合物共结晶、原位蛋白质水解等方法对蛋白质本身进行分子改造,可明显提高其结晶成功率及晶体质量.随着该方面成功案例的不断积累,分子改造技术越来越凸显出其在蛋白质结构解析中的重要作用,特别是对一些难以结晶或提高晶体质量的蛋白质而言,其应用价值更不可忽视.针对近年来分子改造技术在蛋白质结晶中的应用进行了回顾与总结,并展望了其未来的发展.     

用于蛋白质结晶的形核剂研究综述

获取蛋白质晶体是蛋白质三维结构解析、医疗药品生产、自组装纳米体系构建等过程中重要的步骤。例如,利用X射线衍射技术对蛋白质进行三维结构解析时,首先需要通过结晶条件筛选,获得质量较高的蛋白质晶体,进而进行衍射得到蛋白质结构相关信息。蛋白质结晶需要经历从未饱和区经亚稳区至形核区的形核过程以及从形核区到亚稳区的生长成熟过程。在整个蛋白质结晶过程中,形核过程是至关重要的一步。均相形核过程中,结晶体系中各个部分形核概率相同,当蛋白质结晶体系中溶液的过饱和度足够克服形核势垒时,在形核区发生成核,因而在低浓度的结晶溶液体系中,均相形核存在一定的局限性。形核剂的添加使蛋白质晶体异相形核,相较于均相形核其需要克服的阻力小,形核势垒低。因而形核剂的使用对于难结晶蛋白或者起始浓度过低的蛋白质结晶具有重要意义。随着结构生物学的发展,形核剂在蛋白质结晶中的研究仍是结晶方法学领域的热点问题。多孔微球对蛋白质分子的吸附作用有利于无序蛋白质分子团簇的形成,进而促进蛋白质形核。添加多孔微球不但可以增加结晶条件筛选数,也可以提高晶体质量。促进蛋白质分子有序排列的形核剂籽晶的使用,使晶体的形核生长过程始终处于结晶体系溶液浓度较低的状态,而交联的籽晶因为稳定性更高而更有应用前景。新型交互扩散结晶板中,蛋白质结晶体系通过一个较缓慢的交互扩散过程实现蛋白质结晶溶液浓度的变化,并且结晶体系可达到共平衡,因而能显著提高蛋白质晶体结晶条件筛选数和晶体质量:蛋白酶K结晶条件数由39个提升至47个,分辨率由1.66提升至1.54。利用基底材料的一些特性,如静电作用、疏水作用和氢键,可以起到促进蛋白质分子聚集的功能,从而促进形核。本文从物理作用和化学作用两个角度详细总结了形核剂对蛋白质结晶的影响,并展望了该领域的发展前景及研究方向。     

温度控制在蛋白质结晶中的应用

生长蛋白质晶体是X射线衍射技术解析蛋白质分子结构的主要瓶颈问题之一.影响蛋白质结晶的因素很多,其中温度是影响蛋白质结晶的重要因素,但在实践中并未受到广泛重视.总结了恒温和变温对蛋白质可结晶性和晶体质量的影响,发现温度控制既可以获得更多的蛋白质结晶条件,又可以提高蛋白质晶体质量,因此,温度控制是蛋白质结晶工作中应当考虑的重要因素.     

强迫流动环境对蛋白质结晶的影响

生长高质量蛋白质晶体一直是结构生物学领域的瓶颈问题之一。因此,研究蛋白质结晶方法学,提高获得高质量蛋白质晶体的几率,意义重大。强迫流动作为一种特殊的物理环境,对蛋白质溶液形核以及晶体生长过程有着显著的影响。综述了不同的强迫流动环境对蛋白质结晶过程的影响,并对影响机理进行了分析。近期的研究发现,对某些蛋白质而言,强迫流动环境有利于生长尺寸大、质量高的蛋白质晶体。     

牛血清白蛋白Ⅴ的渗透膜结晶研究

采用聚偏氟乙烯中空纤维微孔膜进行牛血清白蛋白Ⅴ的静态渗透膜结晶研究,选择了适宜的沉淀剂和缓冲液,并考察了膜面积和温度对蛋白质膜结晶过程的影响.结果表明,在以pH6.3的磷酸盐为缓冲溶液、氯化钠为沉淀剂的条件下,15℃时用5根中空纤维膜进行膜结晶获得的牛血清白蛋白Ⅴ的晶体质量完美、尺寸大且均匀,适合于X射线衍射分析,证实了膜结晶技术用于制备牛血清白蛋白Ⅴ晶体的可行性.     

渗透膜结晶法结晶木瓜蛋白酶

用中空纤维微孔疏水膜进行木瓜蛋白酶的渗透膜结晶,研究温度和不同pH的缓冲溶液对木瓜蛋白酶静态渗透膜结晶的影响,优化木瓜蛋白酶渗透膜结晶的结晶条件.通过监控结晶溶液中木瓜蛋白酶浓度随时间的变化,考察沉淀剂和添加剂对结晶过程的影响,并对晶体进行尺寸分布分析.结果表明,对于木瓜蛋白酶的渗透膜结晶,在15～32℃下,以pH 4.7的乙酸盐为缓冲溶液,在沉淀剂(硫酸铵和磷酸钠)质量分数4%时,制备出质量较好的木瓜蛋白酶晶体.添加剂PEG600和PEG4000的加入提高了溶剂跨膜通量,改善了晶体质量.     

真空膜结晶法结晶溶菌酶的实验研究

采用聚偏氟乙烯中空纤维微孔膜对真空膜结晶溶菌酶进行了实验研究.考察了膜下游真空度、蛋白质浓度、料液流速、添加剂、pH和离子强度对溶剂跨膜通量及溶菌酶晶体质量的影响.结果表明,真空度应控制在0.015 MPa左右;溶菌酶真空膜结晶的最佳浓度为20 mg/mL;结晶溶液的流速以288μm/s为宜;结晶溶液中加入一定添加剂如丙三醇、DMSO等,可以有效提高溶剂跨膜通量,改善晶体质量;在相同离子强度下,pH值越大膜通量越低,在不同的pH值下可以得到不同形状的晶体.     

超声在工业结晶的应用

超声在结晶过程中的应用,近几年引起了国内外学者的广泛兴趣,主要探讨超声对结晶过程中结晶溶液的诱导期、溶液的一次成核、二次成核、晶体的结晶速率以及晶体的形貌和结构的影响.发现超声可以缩短诱导期,加速晶核的生成.提高晶体的结晶速率,改变晶体的形貌.特别是对产品的影响,可以更好地满足人们对产品的要求.     

衬底温度对Si(111)衬底上MBE异质外延3C-SiC薄膜的影响

利用固源分子束外延(SSMBE)技术, 在Si(111)衬底上异质外延生长3C-SiC单晶薄膜, 通过RHEED、XRD、AFM、XPS等实验方法研究了衬底温度对薄膜结构、形貌和化学组分的影响. 研究结果表明, 1000℃生长的样品具有好的结晶质量和单晶性. 在更高的衬底温度下生长, 会导致大的孔洞形成, 衬底和薄膜间大的热失配使降温过程中薄膜内形成更多位错, 从而使晶体质量变差. 在低衬底温度下生长, 由于偏离理想的化学配比也会导致薄膜的晶体质量降低.     

蛋白质结晶条件的筛选策略研究进展

蛋白质分子的三维结构是生命科学研究中极为重要的信息,X射线单晶衍射技术是目前获得结构信息最主要的手段,但如何筛选到第一个蛋白晶体是该技术必需的第一步,也是制约结构生物学发展的主要瓶颈问题之一。现在一般通过规模筛选的方法从众多的溶液中筛选出可结晶的条件,但是工作量较大,效率也不高。回顾了近年来在提高结晶筛选效率方向取得的成就,主要表现在2个方面:一是在传统结晶方法和试剂基础上发展起来的一系列新的高效的结晶技术和筛选试剂盒;二是从生物学、物理学和化学等角度提出的一些提高结晶筛选效率的新技术,主要包括通过分子工程改造蛋白、提高蛋白溶液的稳定和均一性、导入籽晶、共结晶、改善结晶界面和变温筛选等技术。最后展望了该领域未来的发展趋势。     

Fabrication of photonic crystals on several kinds of semiconductor materials by using focused-ion beam method

In this paper, we introduced the fabrication of photonic crystals on several kinds of semiconductor materials by using focused-ion beam machine, it shows that the method of focused-ion beam can fabricate two-dimensional photonic crystal and photonic crystal device efficiently, and the quality of the fabricated photonic crystal is high. Using the focused-ion beam method, we fabricate photonic crystal wavelength division multiplexer, and its characteristics are analyzed.     

X-ray topography of piezoelectric La(3)Ta(14)Ga(5.5)O(14) crystal grown by Czochralski Method.

We performed synchrotron X-ray topography on a La(3)Ta0(0.5)Ga (5.5)O(14) (LTG) crystal grown by the Czochralski method. Since a synchrotron X-ray source can provide high-energy X-rays, one can detect bulk structures by X-ray topography. LTG is one of the most attractive piezoelectric crystals along with La(3)Ga(5)SiO(14) (LGS) because of its excellent acoustic properties (temperature compensation of acoustic losses). Since LTG single crystals can be grown from a stoichiometric melt, it was expected that single crystals with better quality than the LGS crystal, which cannot be grown from a stoichiometric system but only from a congruent melt, can be obtained. However, 60 keV X-ray topography revealed that the LTG crystal quality was not as high as the LGS crystal quality. The crystal quality of the central region was lower than that of the surrounding region.     

Homogeneous Si0.5Ge0.5 bulk crystal growth as substrates for strained Ge thin films by the traveling liquidus-zone method

Compositionally uniform Si_0.5Ge_0.5 bulk crystals were grown by the traveling liquidus-zone method which we developed for alloy crystal growth. Grown crystals were characterized as substrates for compressive-strained Ge thin films for high mobility p-channels of complementary metal oxide semiconductor transistors. Compositional uniformity was excellent and crystallinity was also excellent for 10 mm diameter crystals. However, crystallinity was degraded for 30 mm diameter crystals although compositional uniformity was excellent. Transmission electron microscope (TEM) observation showed high dislocation density at the interface between a Si seed and a grown crystal due to lattice mismatch. However, the dislocation density decreased as crystal growth proceeded. High quality 30 mm diameter crystals will be grown when the single crystal length is extended judging from TEM results. In this paper, we report on the growth and characterization of Si_0.5Ge_0.5 crystals as substrates for strained Ge thin films.     

X-ray Topography of Piezoelectric La/sub 3/Ta/sub 0.5/Ga/sub 5.5/O/sub 14/ Crystal Grown by Czochralski Method

We performed synchrotron X-ray topography on a La₃Ta_0.5Ga_5.5O₁₄ (LTG) crystal grown by the Czochralski method. Since a synchrotron X-ray source can provide high-energy X-rays, one can detect bulk structures by X-ray topography. LTG is one of the most attractive piezoelectric crystals along with La₃Ga₅SiO₁₄ (LGS) because of its excellent acoustic properties (temperature compensation of acoustic losses). Since LTG single crystals can be grown from a stoichiometric melt, it was expected that single crystals with better quality than the LGS crystal, which cannot be grown from a stoichiometric system but only from a congruent melt, can be obtained. However, 60 keV X-ray topography revealed that the LTG crystal quality was not as high as the LGS crystal quality. The crystal quality of the central region was lower than that of the surrounding region.     

Crucibleless crystal growth and Radioluminescence study of calcium tungstate single crystal fiber

In this article, single phase and high optical quality scheelite calcium tungstate single crystal fibers were grown by using the crucibleless laser heated pedestal growth technique. The as-synthesized calcium tungstate powders used for shaping seed and feed rods were investigated by X-ray diffraction technique. As-grown crystals were studied by Raman spectroscopy and Radioluminescence measurements. The results indicate that in both two cases, calcined powder and single crystal fiber, only the expected scheelite CaWO₄ phase was observed. It was verified large homogeneity in the crystal composition, without the presence of secondary phases. The Radioluminescence spectra of the as-grown single crystal fibers are in agreement with that present in Literature for bulk single crystals, presented a single emission band centered at 420 nm when irradiated with β-rays.     

Free‐Standing 2D Hexagonal Aluminum Nitride Dielectric Crystals for High‐Performance Organic Field‐Effect Transistors

The existence of defects and traps in a transistor plays an adverse role on efficient charge transport. In response to this challenge, extensive research has been conducted on semiconductor crystalline materials in the past decades. However, the development of dielectric crystals for transistors is still in its infancy due to the lack of appropriate dielectric crystalline materials and, most importantly, the crystal morphology required by the gate dielectric layer, which is also crucial for the construction of high‐performance transistor as it can greatly improve the interfacial quality of carrier transport path. Here, a new type of dielectric crystal of hexagonal aluminum nitride (AlN) with the desired 2D morphology of combing thin thickness with large lateral dimension is synthesized. Such a suitable morphology in combination with the outstanding dielectric properties of AlN makes it promising as a gate dielectric for transistors. Furthermore, ultrathin 2,6‐diphenylanthracene molecular crystals with only a few molecular layers can be prepared on AlN crystal via van der Waals epitaxy. As a result, this all‐crystalline system incorporating dielectric and semiconductor crystals greatly enhances the overall performance of a transistor, indicating the importance of minimizing defects and preparing high‐quality semiconductor/dielectric interface in a transistor configuration.     

泡生法生长掺碳钛宝石激光晶体的研究

钛宝石激光晶体在现代高功率激光领域具有重要的应用价值, 但大尺寸、高品质的晶体生长仍是当前钛宝石应用面临的重大难题. 本文研究了泡生法技术生长大尺寸掺碳钛宝石激光晶体, 结果显示, 泡生法生长得到的直径180 mm、30 kg的钛宝石没有出现应力集中的开裂等宏观缺陷现象, 钛离子在晶体中分布均匀接近理论值, 晶体的FOM值达到200. 该研究对低红外残余吸收, 高品质因素、大尺寸钛宝石激光晶体的生长应用具有重要的现实意义.     

A:Al2O3（A=Cr,Fe,Ni）晶体生长及其缺陷研究

报道了A:Al2O3(A=Cr,Fe,Ni)晶体光学浮区法生长工艺,研究了旋转速率、生长速率对晶体质量的影响,制备出了φ6~8 mm、长度为60~80 mm的A:Al2O3晶体.A:Al2O3晶体的生长方向为<001>方向,X射线双晶摇摆曲线表明A:Al2O3晶体具有良好的晶体质量.通过X射线衍射、扫描电镜、偏光显微镜对晶体中的生长缺陷进行了研究,结果表明,A:Al2O3晶体的主要缺陷为小角度晶界、包裹体和溶质尾迹.研究了A:Al2O3晶体的光谱性能,并对A:Al2O3晶体的介电性能进行了测量,室温下1000 kHz时A:Al2O3晶体表现出较高的介电系数εr(12.1~15.7)和较小的介电损耗tanδ(0.0020~0.0002).     

Mica, a potential two-dimensional-crystal gate insulator for organic field-effect transistors

2D mica crystals (with thickness < 100 nm) obtained by mechanical exfoliation are incorporated for the first time into the design of organic thin film field-effect transistor arrays and organic single crystal transistors as a gate insulator. The size of mica crystals could be up to the dimensions of an A4 piece of paper. All devices so fabricated exhibited high mobility and low operating voltage, indicating the high quality of the crystals and the great potential of mica crystals as a flexible, low-cost, transparent insulator for organic electronics.